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Molecular Probes The Handbook

Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4

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Multiplexed Proteomics Technology

Molecular Probes is the leader in the growing field of Multiplexed Proteomics technology — the simultaneous detection of multiple protein targets in a single sample. We have developed a suite of compatible methodologies for the differential staining of specific proteins and the total-protein profile in two or more visually distinguishable colors, producing a more complete picture of the proteome than has ever before been possible. Our state-of-the-art Multiplexed Proteomics technology not only offers the capacity for multicolor staining, but also provides a combination of high sensitivity and simplicity that can streamline protocols and accelerate the pace of research.

Our Multiplexed Proteomics technology relies on a set of protein stains that enable the global detection of differences both in specific protein expression (phosphoproteins, glycoproteins or membrane proteins) and in total-protein expression in 1-D and 2-D polyacrylamide gels or on blots. Our fluorescent and luminescent protein gel stains include:


The Pro-Q Diamond phosphoprotein gel stain, Pro-Q Emerald glycoprotein gel stains and SYPRO Ruby protein gel stain — which we have optimized to complement each other in selectivity, sensitivity and staining protocols — are used in serial detection of phosphoproteins, glycoproteins and total proteins on a single protein sample separated by 1-D or 2-D gel electrophoresis (Figure 9.42). Our Rhinohide polyacrylamide gel strengthener (R33400, R33410; described at the end of this section) greatly improves the strength of any polyacrylamide gel, making it easy to perform these multiple staining procedures without special gel handling. After each staining step, an image of the gel is collected. Once collected, the three images can be overlaid in any combination for analysis of phosphorylation, glycosylation and total-protein expression. Because all three stains are used on the same gel, unambiguous spot matching of phosphoproteins and glycoproteins is made simple by direct comparison with the total-protein profile provided by the SYPRO Ruby protein gel stain. This simultaneous measurement of several variables ensures perfect spatial registration of signals and increases the amount of data that can be collected in a single experiment, leading to more controlled experiments, more accurate data comparisons and fewer ambiguities. When used in combination with the SYPRO Ruby protein gel stain, the Pro-Q Amber transmembrane protein gel stain extends the power of our Multiplexed Proteomics technology to allow selective identification of transmembrane proteins.

For multiplexed protein blot staining, we have developed fluorescent phosphoprotein and glycoprotein stains, as well as several different Western blot stains for detecting specific antibody-tagged proteins or protein modifications (Fluorescence-based Western blot stain kits - Table 9.9):

  • Pro-Q Diamond Phosphoprotein Blot Stain Kit (P33356)
  • Pro-Q Glycoprotein Blot Stain Kits containing Pro-Q Emerald stains (P21857, P21875)
  • Pro-Q Glycoprotein Blot Stain Kit with Griffonia simplicifolia lectin II (GS-II)–based glycoprotein detection (P21872)
  • Amplex Gold Western Blot Stain Kits (A21890, A21891, A21892)
  • Pro-Q Western Blot Stain Kits (P21860, P21861, P21862, P21864, P21865)
  • DyeChrome Western Blot Stain Kits containing either DDAO phosphate (D21881, D21882, D21883) or ELF 39 phosphate (D21884, D21885, D21886)
  • DyeChrome Double Western Blot Stain Kit (D21887), which combines a blue-fluorescent total-protein stain with the immunostaining techniques in our DyeChrome Western Blot Stain Kits and our Amplex Gold Western Blot Stain Kits to yield simultaneous trichromatic detection of two different enzyme-conjugated antibodies on the same blot
  • The BOLD APB chemiluminescent substrate (B21901) for chemiluminescent detection of alkaline phosphatase conjugates on Western blots

The Pro-Q Glycoprotein Blot Stain Kits, Amplex Gold Western Blot Stain Kits and Pro-Q Western Blot Stain Kits can all be paired with our luminescent SYPRO Ruby protein blot stain (S11791) to detect the total-protein profile on the same blot, ensuring accurate registration of protein bands or spots. The DyeChrome Western Blot Stain Kits each provide a compatible fluorescence-based total-protein detection method for counterstaining the entire protein complement on the blot.

Lastly, this section describes several specialized techniques for detecting specific proteins in gels and on blots. Fluorescence technology from Molecular Probes permits the selective detection of oligohistidine fusion proteins in gels or on blots with ease and sensitivity using our Pro-Q Sapphire oligohistidine gel stains (P21876, P21877, P33354) or our Pro-Q Oligohistidine Blot Stain Kits with biotin NTA–based detection (P21878, P21879). We also describe methods for detecting calcium-binding proteins, enzymes, protein functional groups and penicillin-binding proteins, which can potentially be combined with the general protein stains described in Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3 for multiplexed protein staining that is tailored to answer specific research questions.





Figure 9.42 An overview of the Multiplexed Proteomics approach. Images collected after each staining step can be overlaid in any combination for analysis of protein expression, phosphorylation and glycosylation between samples.

Pro-Q Diamond Phosphoprotein Stain for Gels and Blots

Pro-Q Diamond Phosphoprotein Gel Stain and Destain

Molecular Probes' Pro-Q Diamond phosphoprotein gel stain ref is a breakthrough technology that provides a simple, direct method for selectively staining O-linked phosphoproteins in polyacrylamide gels (Figure 9.43). It is ideal for the identification of kinase targets in signal transduction pathways and for phosphoproteomic studies. This proprietary fluorescent stain allows direct, in-gel detection of phosphate groups attached to tyrosine, serine or threonine residues. The Pro-Q Diamond phosphoprotein gel stain can be used with standard SDS-polyacrylamide gels (Figure 9.43) or with 2-D gels (photo) — blotting is not required and there is no need for radioisotopes, phosphoprotein-specific antibodies or Western blot detection reagents. The simple and reliable staining protocol delivers results in as little as 4 to 5 hours. The stain is also compatible with mass spectrometry, allowing meaningful analysis of the phosphorylation state of entire proteomes for the first time. The Pro-Q Diamond phosphoprotein gel stain provides:

  • Simple in-gel detection. Proteins containing phosphate groups attached to tyrosine, serine or threonine residues can be detected directly in either 1-D or 2-D polyacrylamide gels after the gel is fixed, stained and destained; no antibodies are required and no blotting is necessary.
  • Selectivity without radioactivity. The Pro-Q Diamond phosphoprotein gel stain is a fluorescent stain that selectively detects phosphoproteins; radioisotopes are not used and therefore no radioactive waste is generated.
  • Sensitivity. The Pro-Q Diamond phosphoprotein gel stain allows the detection of as little as 1–16 ng of phosphoprotein per band, depending on the phosphorylation level of the protein.
  • Quantitation. The Pro-Q Diamond signal for individual phosphoproteins is linear over three orders of magnitude and correlates with the number of phosphate groups (Figure 9.45).
  • Compatibility. Pro-Q Diamond gel stain (excitation/emission maxima ~555/580 nm) is compatible with a visible-light–scanning instrument, a visible-light transilluminator or (with reduced sensitivity) a 300 nm transilluminator, as well as with mass spectrometry analysis (Figure 9.46).
  • Multiplexing capability. Pro-Q Diamond gel stain can be used with SYPRO Ruby protein gel stain (Figure 9.43) and Pro-Q Emerald glycoprotein gel stain on the same gel for multiparameter staining.

The Pro-Q Diamond phosphoprotein gel stain (Pro-Q Diamond gel stain reagents and kits - Table 9.6) is supplied ready-to-use in three different sizes: a 200 mL size (P33301) suitable for staining approximately four minigels; a 1 L size (P33300) suitable for staining approximately 20 minigels or two large-format gels, e.g., 2-D gels; and a 5 L bulk-packaging size (P33302). In addition, we offer Pro-Q Diamond Phosphoprotein Gel Staining Kits (MPP33300, MPP33301, MPP33302) that include both the Pro-Q Diamond gel stain and the PeppermintStick phosphoprotein molecular weight standards (see below). All products are accompanied by a simple and reliable staining and destaining protocol (Product Information Sheet) that delivers results in as little as four to five hours. For convenient destaining, we also offer the Pro-Q Diamond phosphoprotein gel destaining solution as a ready-to-use solution in either a 1 L (P33310) or 5 L (P33311) size.





Figure 9.43 Selectivity of the Pro-Q Diamond phosphoprotein gel stain (P33300, P33301, P33302) for phosphoproteins. A polyacrylamide gel containing various proteins was stained with Pro-Q Diamond phosphoprotein gel stain (panel A) followed by SYPRO Ruby protein gel stain (panel B). This gel shows a nonphosphorylated protein, lysozyme (lanes 3 and 4), as well as several phosphoproteins, α-casein (lanes 1 and 2), ovalbumin (lanes 5 and 6) and pepsin (lanes 7 and 8), before (even lanes) and after (odd lanes) treatment with phosphatases. Loss of Pro-Q Diamond staining indicates loss of all phosphates from pepsin, partial loss of phosphates from α-casein and ovalbumin and no change in the nonphosphorylated protein lysozyme.




Figure 9.45 Sensitivity and linear range of Pro-Q Diamond phosphoprotein gel stain (P33300, P33301, P33302). Six different proteins were serially diluted and run on separate SDS-polyacrylamide gels, which were then stained with Pro-Q Diamond phosphoprotein gel stain. The images were documented on a fluorescence imager, and the fluorescence emission from each band was quantitated. The number of known phosphate groups on each protein is indicated in the Figure .egend. A) Fluorescence emission of the band, plotted as a function of protein amount, in nanograms. B) Magnification of data points in the highlighted box in panel A. C) The fluorescence emission of the band, plotted as a function of picomoles of protein. D) Magnification of the data points in the highlighted box in panel C. E) The slope of the line for each protein in panel C, plotted against the known number of phosphates per protein.




Figure 9.46 Fluorescence excitation and emission spectra of the Pro-Q Diamond phosphoprotein gel stain (P33300, P33301, P33302).


Multiplexed Proteomics Kits for Phosphoprotein and Total-Protein Gel Staining

When used together, the Pro-Q Diamond phosphoprotein gel stain and the SYPRO Ruby protein gel stain (S12000, S12001, S21900; Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3) make a powerful combination for proteome analysis. The SYPRO Ruby dye is a total-protein stain that, like the Pro-Q Diamond gel stain, is quantitative over three orders of magnitude. Determining the ratio of the Pro-Q Diamond dye to SYPRO Ruby dye signal intensities for each band or spot thus provides a measure of the phosphorylation level normalized to the total amount of protein. Using both stains in combination makes it possible to distinguish a low amount of a highly phosphorylated protein from a higher amount of a less phosphorylated protein. To make this staining more convenient and economical, we offer the Multiplexed Proteomics Kit #1 with 200 mL of the Pro-Q Diamond phosphoprotein gel stain and 200 mL of the SYPRO Ruby protein gel stain (M33306), the Multiplexed Proteomics Kit #2 with 1 L of each stain (M33305) and the Multiplexed Proteomics Phosphoprotein Gel Stain Kits (MPM33305, MPM33306), which include the Pro-Q Diamond phosphoprotein gel stain, SYPRO Ruby protein gel stain and PeppermintStick phosphoprotein molecular weight standards (see below); Pro-Q Diamond gel stain reagents and kits - Table 9.6 summarizes all of our Pro-Q Diamond gel stain reagents and kits.

Pro-Q Diamond Phosphoprotein Blot Stain Kit

The Pro-Q Diamond Phosphoprotein Blot Stain Kit (P33356) provides a simple and quick method for directly detecting phosphoproteins on poly(vinylidene difluoride) (PVDF) (Figure 9.47) or nitrocellulose membranes without the use of radioactivity or antibodies. As with the gel stain described above, the Pro-Q Diamond phosphoprotein blot stain detects phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins, independent of the sequence context of the phosphorylated amino acid residue. Thus, the native phosphorylation levels of proteins from a variety of sources, including tissue specimens and body fluids, can be analyzed. Protein samples are separated by 1-D or 2-D gel electrophoresis, electroblotted to the membrane, stained and destained using a protocol similar to that typically performed with amido black or Ponceau S staining of total-protein profiles on membranes. After staining, gels are simply imaged using any of a variety of laser scanners, xenon-arc lamp–based scanners or CCD-based imaging devices employing UV transilluminators; the excitation/emission maxima of the Pro-Q Diamond phosphoprotein blot stain are ~555/580 nm. The limits of detection for the stain on PVDF membrane blots are typically 8–16 ng of phosphoprotein, with a linear dynamic range of approximately 15-fold. The sensitivity of the Pro-Q Diamond phosphoprotein blot stain is decreased when using nitrocellulose blots. Each Pro-Q Diamond Phosphoprotein Blot Stain Kit provides:


Sufficient reagents are provided for staining ~20 minigel electroblots.

The Pro-Q Diamond phosphoprotein blot stain binds noncovalently to phosphoproteins and is thus fully compatible with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) and Edman sequencing. Furthermore, the Pro-Q Diamond phosphoprotein blot stain is compatible with the standard colorimetric, fluorometric and chemiluminescent detection techniques employed in immunoblotting. This phosphoprotein blot stain may be used in conjunction with the SYPRO Ruby protein blot stain, a total-protein stain that is quantitative over two orders of magnitude on blots. Using the SYPRO and Pro-Q Diamond blot stains in combination makes it possible to distinguish a low amount of a highly phosphorylated protein from a higher amount of a less phosphorylated protein.

PeppermintStick Phosphoprotein Molecular Weight Standards

PeppermintStick phosphoprotein molecular weight standards are a mixture of phosphorylated and nonphosphorylated proteins with molecular weights from 14,400 to 116,250 daltons (PeppermintStick Phosphoprotein Molecular Weight Standards). Separation by polyacrylamide gel electrophoresis resolves this mixture into two phosphorylated and four nonphosphorylated protein bands (photo). These standards serve both as molecular weight markers and as positive and negative controls for our Pro-Q Diamond phosphoprotein gel stain and other methods that detect phosphorylated proteins. We offer two different unit sizes of the PeppermintStick phosphoprotein molecular weight standards: a 40 µL unit size sufficient for 20–40 gel lanes (P27167) and a 400 µL unit size sufficient for 200–400 gel lanes (P33350).

Phosphopeptide Standard Mixture

Formulated especially for MALDI-MS, the phosphopeptide standard mixture (P33357) contains equimolar amounts of three unphosphorylated and four phosphorylated peptides, ranging in mass from 1047 to 2192 and representing phosphoserine (pS), phosphothreonine (pT) and phosphotyrosine (pY) monophosphopeptides, as well as a peptide containing both pT and pY. This mixture is ideal for use as an internal or external control for LC/MS, MALDI analysis or β-elimination reactions.

Pro-Q Diamond Phosphoprotein Enrichment Kits

The Pro-Q Diamond Phosphoprotein Enrichment Kit (P33358) enables efficient, nonradioactive isolation of phosphoproteins from complex cellular extracts. This kit provides resin, reagents and columns designed to isolate phosphoproteins from 0.5–1.0 mg of total cellular protein per column. The column bed volume can be easily scaled up or down depending on the amount of available starting material. The phosphoprotein-binding properties of the resin allow efficient capture of both native and denatured proteins. Therefore, cell or tissue samples can be denatured in lysis buffers and stored in the freezer prior to the phosphoprotein enrichment procedure. Each Pro-Q Diamond Phosphoprotein Enrichment Kit contains:

Phosphoprotein Enrichment Module

  • Resin (50% v/v slurry)
  • Disposable 2 mL columns, 10 columns
  • Lysis buffer
  • Wash buffer
  • Elution buffer
  • Vivaspin filtration concentrators with 10 kDa cutoff polyethersulfone membrane, 10 concentrators

Protease Inhibitor and Endonuclease Module

  • Protease inhibitor
  • Endonuclease

Protocols for both undenatured and denatured lysates are provided, and these procedures can be completed in approximately three hours. For added convenience, the Pro-Q Diamond Phosphoprotein Enrichment and Detection Kit (P33359) provides all the reagents in the Pro-Q Diamond Phosphoprotein Enrichment Kit, as well as Pro-Q Diamond phosphoprotein gel stain and PeppermintStick phosphoprotein molecular weight markers for detecting phosphoproteins on SDS-polyacrylamide gels.

Other Reagents for Phosphoproteomics

The Captivate Microscale Phosphopeptide Isolation Kit (C33355), described in detail in Quantitation and Selective Purification of Proteins in Solution - Section 9.2, provides a highly selective and sensitive method for isolating phosphopeptides from complex solutions. This technology is ideal for isolating phosphorylated peptides as a front-end fractionation step prior to liquid chromatography– and mass spectrometry–based proteomics systems.

The Antibody Beacon Tyrosine Kinase Assay Kit (A35725), described in detail in Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3, provides a simple yet robust solution assay for real-time monitoring of tyrosine kinase activity and the effectiveness of potential inhibitors and modulators. The key to this tyrosine kinase assay is a small-molecule tracer ligand labeled with our bright green-fluorescent Oregon Green 488 dye. When an anti-phosphotyrosine antibody binds this tracer ligand to form the Antibody Beacon detection complex, the fluorescence of the Oregon Green 488 dye is efficiently quenched. In the presence of a phosphotyrosine-containing peptide, however, this Antibody Beacon detection complex is rapidly disrupted, releasing the tracer ligand and relieving its antibody-induced quenching. Upon its displacement by a phosphotyrosine residue, the Oregon Green 488 dye-labeled tracer ligand exhibits an approximately fourfold enhancement in its fluorescence, enabling the detection of as little as 50 nM phosphotyrosine-containing peptide with excellent signal-to-background discrimination.

Pro-Q Glycoprotein Stain Kits for Gels and for Blots

Glycoproteins play important roles as cell-surface markers and in cell adhesion, immune recognition and inflammation reactions.ref To facilitate research on glycoproteins, Molecular Probes has introduced the Pro-Q Glycoprotein Stain Kits for Gels and for Blots, which provide unsurpassed sensitivity, linearity and ease of use for selective detection of glycoproteins.

Pro-Q Emerald Glycoprotein Stain Kits for Gels and for Blots

Our Pro-Q Emerald 300 and Pro-Q Emerald 488 Glycoprotein Stain Kits (Pro-Q Emerald glycoprotein stain kits for gels and for blots - Table 9.7) provide the most advanced reagents known for detecting glycoproteins in gels and on blots.ref These stains are easier to use and more sensitive than any other glycoprotein staining technique (Comparison of various commercially available glycoprotein stain kits - Table 9.8). The Pro-Q Emerald glycoprotein stains react with periodate-oxidized carbohydrate groups, creating a bright green-fluorescent signal on glycoproteins (Figure 9.49). The staining procedure requires only three steps: fixation, oxidation and staining — no reduction step is required (Figure 9.50). Depending on the nature and degree of glycosylation, the Pro-Q Emerald 300 stain allows the detection of as little as 1 ng of a glycoprotein per band in gels (4 ng/band with the Pro-Q Emerald 488 stain), making these stains about 50 times more sensitive than the standard periodic acid–Schiff base method using acidic fuchsin dye. Blot staining is not quite as sensitive (2–18 ng of a glycoprotein per band can be detected) and is more time consuming, but provides an opportunity to combine glycoprotein staining with immunostaining or other blot-based detection techniques. The Pro-Q Emerald 300 stain is best visualized using 300 nm UV illumination, whereas the Pro-Q Emerald 488 stain is best visualized using visible light with wavelengths near its 510 nm excitation maximum. The Pro-Q Emerald dye is also used as the detection reagent in our Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495), which is described in Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2 (Figure 3.19, Figure 3.20, Figure 3.21).




Figure 3.19 Lipopolysaccharide staining with the Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit. Lipopolysaccharides (LPS) were electrophoresed through a 13% acrylamide gel and stained using the Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495). From left to right, the lanes contain: CandyCane glycoprotein molecular weight standards (~250 ng/band), blank, 4, 1 and 0.25 µg of LPS from Escherichia coli smooth serotype 055:B5 and 4, 1 and 0.25 µg of LPS from E. coli rough mutant EH100 (Ra mutant).


Figure 3.20 Characterization of lipopolysaccharides. Lipopolysaccharides (LPS) from Escherichia coli smooth serotype 055:B5 were loaded onto a 13% polyacrylamide gel. Following electrophoresis, the gel was stained using the Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495), and the fluorescence was measured for the lane. A plot of fluorescence signal versus the relative distance from the dye front shows a characteristic laddering profile for smooth-type LPS.



Figure 3.21 Linearity of the Pro-Q Emerald 300 stain for lipopolysaccharide (LPS) detection. A dilution series of lipopolysaccharides from Escherichia coli smooth serotype 055:B5 was loaded onto a 13% polyacrylamide gel. Following electrophoresis, the gel was stained using the Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495) and the same band from each lane was quantitated using a CCD camera. A plot of the fluorescence intensity versus the mass of LPS loaded shows a linear range over two orders of magnitude.




Figure 9.49 Detecting glycoproteins with Pro-Q Emerald glycoprotein detection reagents. Oxidation with periodic acid converts cis-glycols to dialdehydes, which can then react with the hydrazide-based Pro-Q Emerald reagents to form a covalent bond.



Figure 9.50 Detecting glycoproteins with the Pro-Q Emerald 300 glycoprotein detection reagents requires only three steps: fixation, periodate oxidation of the cis-glycols to dialdehydes, and incubation with the Pro-Q Emerald 300 glycoprotein detection reagent (P21855, P21857). No destaining step is required.



The Pro-Q Emerald glycoprotein stains can be combined with general protein stains for dichromatic detection of glycoproteins and total proteins in gels and on blots, making it much easier to identify the location of the glycoproteins in the total-protein profile (Figure 9.51, Figure 9.52, Figure 9.53, photo). The easy-to-use Patented SYPRO Ruby protein gel and blot stains (described in Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3) provide the same sensitivity as silver staining (gels) or colloidal gold staining (blots) but, unlike these chromogenic techniques, do not require formaldehyde or glutaraldehyde, which can produce false positive responses when glycoproteins are stained. These total-protein stains make it possible to visualize the entire protein complement of a sample and to thus identify contaminating proteins, to compare stained proteins to molecular weight standards and to provide a control for protease contamination in glycosidase mobility-shift experiments. The SYPRO Ruby protein blot stain is additionally useful for assessing the efficiency of protein transfer to a blot (Figure 9.53), which is especially important when working with glycoproteins, because they often transfer poorly to blotting membranes. Proteins labeled with the SYPRO Ruby total-protein stains exhibit red-orange fluorescence when excited with either a 300 nm UV light source or a laser scanner with a 473, 488 or 532 nm laser light source.

The Pro-Q Emerald Glycoprotein Stain Kits also include our exclusive CandyCane molecular weight standards, a mixture of glycosylated and nonglycosylated proteins that, when separated by electrophoresis, provide alternating positive and negative controls (Figure 9.55). The CandyCane molecular weight standards are also available separately (C21852, CandyCane Glycoprotein Molecular Weight Standards). In addition, we offer a Pro-Q Emerald 300 Glycoprotein Gel Stain Kit (P21855) that includes our SYPRO Ruby protein gel stain for detecting total proteins. The Pro-Q Emerald Glycoprotein Stain Kits for gels and blots contain:


Each kit provides sufficient materials to stain approximately ten 8 cm × 10 cm gels or blots.


Figure 9.51 Multiplexed analysis of normal liver cells (left) and liver tumor cells (right). Gels are stained for total protein and glycoproteins using SYPRO Ruby protein gel stain (S12000, S12001, S21900) and a Pro-Q Emerald glycoprotein stain (P21855, P21857, P21875), respectively. Individual gel images can then be digitally overlaid, matched and analyzed for changes in protein expression and glycosylation.




Figure 9.52 Mobility-shift gel assay using deglycosylating enzymes, stained with the SYPRO Ruby protein gel stain (top, S12000, S12001, S21900) and Pro-Q Emerald 300 glycoprotein stain (bottom). The glycoproteins α1-acidic glycoprotein, fetuin and horseradish peroxidase (HRP) are shown before (lanes 2, 4 and 6, respectively) and after (lanes 3, 5 and 7, respectively) glycosidase treatment. Glycosidase treatment resulted in a mobility shift and loss of green-fluorescent Pro-Q Emerald 300 staining for α1-acidic glycoprotein and fetuin, indicating that the carbohydrate groups had been cleaved off. HRP, which contains an α-(1,3)-fucosylated asparagine GlcNac-linkage that is resistant to many glycosidases, showed neither a mobility shift nor a loss of green-fluorescent Pro-Q Emerald 300 staining. Thus, use of the Pro-Q Emerald 300 Glycoprotein Stain Kits (P21855, P21857, M33307) identifies glycoproteins that are not susceptible to the glycosidases used in the assay, providing important structural information about the glycoprotein's carbohydrate moiety.




Figure 9.53 Staining glycoproteins and the total protein profile on blots using the Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain Kit (P21857). A twofold dilution series of the CandyCane glycoprotein molecular weight standards (C21852) was run an SDS-polyacrylamide gel and blotted onto a PVDF membrane. The blot was first stained with the SYPRO Ruby protein blot stain (S11791) to detect the total protein profile (left). After documentation of the signal, the blot was stained with the Pro-Q Emerald 300 glycoprotein stain (right) provided in the Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain Kit.



Figure 9.55 Glycosylated and nonglycosylated proteins in the CandyCane glycoprotein molecular weight standards (C21852). The standards were electrophoresed through two identical 13% polyacrylamide gels. Both lanes contain ~0.5 µg of protein in each band. The left lane was stained with our SYPRO Ruby protein gel stain (S12000, S12001, S21900) to detect all eight marker proteins. The right lane was stained using the reagents in the Pro-Q Emerald 300 Glycoprotein Gel Stain Kit (P21855).


Multiplexed Proteomics Kit for Glycoprotein and Total-Protein Gel Staining

When used together, the Pro-Q Emerald 300 glycoprotein gel stain and the SYPRO Ruby protein gel stain (S12000, S12001, S21900; Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3) make a powerful combination for proteome analysis (Figure 9.51). Determining the ratio of the Pro-Q Emerald dye to SYPRO Ruby dye signal intensities for each band or spot provides a measure of the glycosylation level normalized to the total amount of protein. Using both stains in combination makes it possible to distinguish a lightly glycosylated, high-abundance protein from a heavily glycosylated, low-abundance protein. To make this staining more convenient and economical, we offer the Multiplexed Proteomics Glycoprotein Gel Stain Kit with 1 L of our Pro-Q Emerald 300 glycoprotein gel stain and 1 L of our SYPRO Ruby protein gel stain (M33307).

Pro-Q Glycoprotein Blot Stain Kit with Lectin-Based Detection

Our lectin-based Pro-Q Glycoprotein Blot Stain Kit provides high-sensitivity detection of specific sugar residues in glycoproteins. Pro-Q Glycoprotein Blot Stain Kit #5 (P21872) is supplied with Griffonia simplicifolia lectin II (GS-II), which recognizes terminal nonreducing N-acetylglucosamine residues. This kit uses an alkaline phosphatase conjugate of the lectin as a convenient means of selectively detecting the corresponding glycoproteins on PVDF or nitrocellulose membranes, providing valuable information about the structure of glycoproteins in an experimental sample. The techniques in this Pro-Q Glycoprotein Kit can also be applied to detect terminal α-mannopyranosyl and α-glucopyranosyl residues (that do not contain tri- or tetra-antennary structures) by using an alkaline phosphatase conjugate of concanavalin A (Con A), as well as to detect N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues by using an alkaline phosphatase conjugate of wheat germ agglutinin (WGA).

The detection procedure in this kit is similar to that of Western blotting and uses a fluorogenic alkaline phosphatase substrate, DDAO phosphate, for the final detection step. After the proteins are separated on a polyacrylamide gel, they are blotted onto a membrane and incubated with the lectin–alkaline phosphatase conjugate. The alkaline phosphatase enzyme is then detected using DDAO phosphate, which is rapidly converted to the long-wavelength, red-fluorescent product DDAO (absorbance maxima ~275 nm and ~646 nm, emission maximum ~659 nm) (Figure 10.7). The enzymatic reaction greatly amplifies the signal, making it possible to detect as little as 15 ng of a glycoprotein, depending on the degree and nature of glycosylation. Sensitivity of the DDAO phosphate–based detection technique rivals ECL chemiluminescence detection, but because DDAO phosphate–based detection produces a stable fluorescent product, there is no need to perform the reaction in a darkroom or to expose the blot to X-ray film. Additionally, unlike transient chemiluminescent signals, the red-fluorescent DDAO signal can be imaged several times and is stable indefinitely on dried blots.

For fluorescent detection of total proteins, this Pro-Q Glycoprotein Blot Stain Kit can be used in conjunction with our highly sensitive SYPRO Ruby protein blot stain (S11791). When used prior to glycoprotein detection, SYPRO Ruby protein blot stain makes it possible to assess the level of protein transfer to the blot, to compare stained proteins with molecular weight markers and to identify contaminating proteins in the sample. SYPRO Ruby protein blot stain is particularly useful for blots of 2-D gels where total-protein staining makes it easier to localize stained glycoproteins in the complex protein pattern. Both the enzymatic hydrolysis product of DDAO phosphate (DDAO) and the SYPRO Ruby blot stain can be easily visualized using either UV epi-illumination or a laser scanner. Pro-Q Glycoprotein Blot Stain Kit #5 also provides a sample of our CandyCane glycoprotein molecular weight standards, which include eight glycoproteins, five of which are recognized by Con A or WGA (Figure 9.55).

Each Pro-Q Glycoprotein Blot Stain Kit (P21872) contains:

  • An alkaline phosphatase conjugate of GS-II lectin
  • DDAO phosphate
  • Dimethylformamide to dissolve the DDAO phosphate
  • CandyCane glycoprotein molecular weight standards
  • Detailed protocols for staining and detection with GS-II (Pro-Q(R) Glycoprotein Blot Stain Kit with GS-II)

Each kit contains sufficient reagents for staining 10–20 minigel blots (8 cm × 10 cm). DDAO phosphate (D6487, Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3), SYPRO Ruby protein blot stain (S11791) and CandyCane molecular weight standards (C21852) are also available separately.




Figure 10.7 Normalized absorption and fluorescence emission spectra of DDAO, which is formed by alkaline phosphatase–mediated hydrolysis of DDAO phosphate (D6487).

CandyCane Glycoprotein Molecular Weight Standards

CandyCane glycoprotein molecular weight standards (C21852) contain a mixture of glycosylated and nonglycosylated proteins with molecular weights from 14,000 to 180,000 daltons (CandyCane Glycoprotein Molecular Weight Standards). When separated by polyacrylamide gel electrophoresis, the standards appear as alternating bands corresponding to glycosylated and nonglycosylated proteins (Figure 9.55). Thus, these standards serve both as molecular weight markers and as positive and negative controls for methods that detect glycosylated proteins, such as those provided in our Pro-Q Emerald Glycoprotein Stain Kits (see above).

Pro-Q Amber Transmembrane Protein Gel Stain

The Multiplexed Proteomics Transmembrane Protein Gel Stain Kit (M33308) provides two fluorescent gel stains — the Pro-Q Amber transmembrane protein gel stain (excitation/emission maxima ~470/570 nm), which is selective for transmembrane proteins in gels, and the SYPRO Ruby protein gel stain (excitation/emission maxima ~280, 450/610 nm), which stains all proteins. Once proteins are separated by SDS-polyacrylamide gel electrophoresis, the gel is simply fixed, stained with the Pro-Q Amber reagent, washed and imaged using a laser scanner. Once the transmembrane proteins are visualized, the gel is then stained with SYPRO Ruby dye to detect the total-protein profile and normalize the Pro-Q Amber signals. Thus, after sequentially staining and imaging a gel, transmembrane proteins can be discriminated from nontransmembrane proteins. With the Pro-Q Amber stain, as little as 10 ng of bacteriorhodopsin, a seven-domain transmembrane protein, can be detected. Moreover, the staining intensity is linear over more than two orders of magnitude, and the staining intensity of bacteriorhodopsin is at least 20 times greater than that of carbonic anhydrase, a nontransmembrane protein (Figure 9.56). The Pro-Q Amber stain has been tested with a number of proteins, including those known to have hydrophobic transmembrane α-helices and those with none, and has been found to preferentially stain proteins with two or more transmembrane domains (Figure 9.57). Each Multiplexed Proteomics Transmembrane Protein Gel Stain Kit contains:


Each kit provides sufficient materials to stain approximately ten 0.5–1 mm thick, 8 cm × 10 cm minigels. The Pro-Q Amber stain is not recommended for staining proteins in 2-D, IEF or nondenaturing gels and is not suitable for staining proteins on blots.




Figure 9.56 Sensitivity of the Pro-Q Amber transmembrane protein gel stain. An SDS-polyacrylamide gel containing a twofold dilution series of the transmembrane protein standards was stained with the Pro-Q Amber transmembrane protein gel stain (top) and subsequently with the SYPRO Ruby protein gel stain (bottom), both of which are provided in the Multiplexed Proteomics Transmembrane Protein Gel Stain Kit (M33308). Lane 1 contains 250 ng of each marker in the broad-range molecular weight markers; lanes 2–11, twofold dilution series of the protein standards provided in the kit, starting with 2 µg carbonic anhydrase and 1.4 µg bacteriorhodopsin in lane 2; lane 12, 250 ng bovine serum albumin (BSA). Carbonic anhydrase (upper band) is a nontransmembrane protein, and bacteriorhodopsin (lower band) is a seven-domain transmembrane protein. The numbers at the left indicate the molecular weights of the markers. The images were acquired using a 473 nm laser–based gel scanner with a 520 nm bandpass filter for the detection of Pro-Q Amber signal and a 580 nm bandpass filter for detection of the SYPRO Ruby signal.



Figure 9.57 Ratio of the Pro-Q Amber transmembrane gel stain signal to the SYPRO Ruby gel stain signal for various transmembrane and nontransmembrane proteins. Proteins were separated by SDS-PAGE, stained with the Pro-Q Amber transmembrane protein gel stain (provided in the Multiplexed Proteomics Transmembrane Protein Gel Stain Kit, M33308) and imaged. The gel was then stained with the SYPRO Ruby protein gel stain (also provided in the kit) and imaged again. The intensity of each band was measured, and the ratio of the two signals was plotted as a bar graph. Proteins tested were as follows: A, bacteriorhodopsin; B, ATP synthase F0 a subunit; C, ATP synthase F0 c subunit; D, ATP synthase F0 b subunit; E, glycophorin; F, porin; G, zein; H, carbonic anhydrase; I, ATP synthase F1 α subunit; J, ATP synthase F1 β subunit; K, ATP synthase F1 γ subunit; L, ATP synthase F1 δ subunit; M, ATP synthase F1 ε subunit; N, myosin; O, β-galactosidase; P, phosphorylase b; Q, bovine serum albumin (BSA); R, ovalbumin; S, soybean trypsin inhibitor; T, lysozyme; and U, aprotinin. The numbers along the x-axis indicate the number of α-helical transmembrane domains present in the corresponding protein; the asterisk denotes a 16-strand anti-parallel β-sheet transmembrane domain.

Western Blot Stain Kits and Reagents

The Western blot immunodetection technique provides a powerful method for detecting a protein or proteins of interest on a PVDF or nitrocellulose membrane. The proteins on the blot are typically incubated with a primary antibody against the protein of interest, followed by either an enzyme-labeled secondary antibody or a biotinylated secondary antibody in conjunction with an enzyme-labeled streptavidin. Finally, presence of the enzyme is detected using chromogenic, fluorogenic or chemiluminescent enzyme substrates. Specific proteins and total proteins are difficult to detect on the same blot using conventional chromogenic stains, which has complicated the assessment of protein transfer efficiency, the identification of contaminating proteins and the localization of an immunostained protein in electroblots of 2-D gels. The development of fluorogenic enzyme substrates and luminescent protein stains solves this problem by making it possible to visualize total proteins and specific proteins on the same blot. The Amplex Gold, Pro-Q and DyeChrome Western Blot Stain Kits use fluorogenic immunostains that can be combined with our proprietary luminescent total-protein detection technologies to make it easy to routinely obtain this valuable information without running duplicate gels. Fluorescent protein detection methods offer high sensitivity, streamlined procedures and the opportunity for multicolor labeling. In the development of these products, we have emphasized reagents that provide a combination of selective and general detection of multiple protein targets by well-resolved dichromatic or polychromatic staining technology.

Amplex Gold Western Blot Stain Kits

Amplex Gold reagent is a fluorogenic horseradish peroxidase (HRP) substrate that provides a sensitive fluorescence-based detection method for Western blots. In the presence of the enzyme, this nonfluorescent substrate is converted to a golden-yellow–fluorescent product (photo). The Amplex Gold reagent is simple to use, producing a fluorescent signal at the reaction site within minutes. The reaction does not require addition of hydrogen peroxide. The signal amplification of the enzymatic reaction allows detection of as little as 1–3 ng of a protein per band, depending on the antibodies used. The signal is stable indefinitely and can be documented using UV epi-illumination and a Polaroid camera; use of the inexpensive Amplex Gold photographic filter (A24772, Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3) produces optimal sensitivity. The signal can also be documented using a laser scanner. Scanners using light sources near the excitation maxima for the Amplex Gold peroxidation product (~515 nm) provide the highest sensitivity. The Amplex Gold Western Blot Stain Kits provide the Amplex Gold reagent in combination with either goat anti–mouse IgG antibody, goat anti–rabbit IgG antibody or streptavidin (see Fluorescence-based Western blot stain kits - Table 9.9). These kits can be used in combination with the SYPRO Ruby protein blot stain (S11791, Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3) for detecting the total-protein profile on the same blot.

Each Amplex Gold Western Blot Stain Kit contains the following reagents, which are sufficient to stain ~20 minigel blots (6 cm × 9 cm):

  • Amplex Gold reagent (10 vials)
  • Solvent for Amplex Gold reagent
  • Reaction buffer
  • Horseradish peroxidase conjugate of goat anti–mouse IgG antibody (in Kit #1, A21890), goat anti–rabbit IgG antibody (in Kit #2, A21891) or streptavidin (in Kit #3, A21892)
  • A detailed protocol (Amplex(R) Gold Western Blot Stain Kits)

Pro-Q Western Blot Stain Kits

Our Pro-Q Western Blot Stain Kits (Fluorescence-based Western blot stain kits - Table 9.9) use the fluorogenic substrate DDAO phosphate for simple and rapid detection of an antibody or streptavidin conjugated to alkaline phosphatase (Figure 9.59). DDAO phosphate is a remarkable reagent that provides very rapid and highly sensitive fluorescence detection of alkaline phosphatase conjugates. Alkaline phosphatase rapidly converts DDAO phosphate to the long-wavelength, red-fluorescent product, DDAO (Figure 9.60, Figure 10.7). The signal amplification of the enzymatic reaction allows detection of as little as 1–3 ng of a protein per band, depending on the antibodies used. The sensitivity rivals that of chemiluminescence-based techniques, but because it results in a stable fluorescent product, there is no need to perform the reactions in a darkroom or to incubate the blots with X-ray film. Furthermore, the fluorescent signals, unlike transient chemiluminescent signals, can be imaged several times and are stable indefinitely on dried blots.

Our Pro-Q Western Blot Stain Kits include:

  • DDAO phosphate substrate with an appropriate solvent
  • Alkaline phosphatase conjugate of either goat anti–mouse IgG antibody (in Kit #2, P21860), goat anti–rabbit IgG antibody (in Kit #3, P21864 and Kit #4, P21861) or streptavidin (in Kit #5, P21865 and Kit #6, P21862)
  • SYPRO Ruby protein blot stain (only in Kits #2, #4 and #6; P21860, P21861, P21862)
  • Detailed protocols for total and specific protein detection (Product Information Sheet)

Kits #2, #4 and #6 also include the SYPRO Ruby protein blot stain for highly sensitive detection of total protein on the blot before immunostaining, as described in detail in Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3. Much more sensitive than Ponceau S, amido black or Coomassie brilliant blue, and fully compatible with immunodetection techniques, this brilliant red-orange–luminescent stain makes it easy to routinely obtain valuable information about the total-protein complement of the sample. The DDAO and SYPRO Ruby signals can both be visualized using either UV epi-illumination or a laser scanner.

Each Pro-Q Western Blot Stain Kit contains sufficient materials to stain approximately 10–20 minigel blots (8 cm × 10 cm).





Figure 9.59 Protein detection with the Pro-Q Western Blot Stain Kit #2 (P21860). Samples of protein molecular weight standards containing decreasing amounts of α-tubulin were run on an SDS-polyacrylamide gel, blotted onto a PVDF membrane and stained with the SYPRO Ruby protein blot stain (top). After staining, the blot was incubated with a mouse monoclonal anti–α-tubulin antibody (not included in the kit, A11126), followed by the alkaline phosphatase conjugate of goat anti–mouse IgG antibody. The enzymatic activity was detected using DDAO phosphate and imaged under UV epi-illumination using the Fluor-S MAX MultiImager documentation system (Bio-Rad Laboratories) (bottom).




Figure 9.60 Schematic diagram of Western blot immunodetection with DDAO phosphate (D6487), a component of the DyeChrome Western Blot Stain Kits #1, #2 and #3 (D21881, D21882, D21883; Fluorescence-based Western blot stain kits - Table 9.9).


DyeChrome Western Blot Stain Kits

Our DyeChrome Western Blot Stain Kits (Fluorescence-based Western blot stain kits - Table 9.9) use a fluorogenic alkaline phosphatase conjugate of a secondary antibody or streptavidin and a fluorogenic alkaline phosphatase substrate for immunodetection of specific proteins in combination with an amine-reactive BODIPY dye to detect all proteins on a blot in a contrasting fluorescent color ref (Figure 9.61).




Figure 9.61 Schematic diagram of Western blot immunodetection with BODIPY TR-X succinimidyl ester and ELF 39 phosphate, which are components of our DyeChrome Western Blot Stain Kits #4, #5 and #6 (D21884, D21885, D21886; Fluorescence-based Western blot stain kits - Table 9.9).


The DyeChrome Western Blot Stain Kits include a novel method of staining total proteins on blots using two of our proprietary amine-reactive BODIPY dyes. The reactive dye forms a permanent covalent bond with proteins that lasts through subsequent immunostaining.ref This staining technique makes it possible to perform simultaneous two-color labeling, with both total protein and immunostained proteins visible at the same time on the same blot (photo, Figure 9.63). BODIPY dye–based staining of the total-protein profile is rapid, simple and highly sensitive — a combination of traits not found in conventional chromophoric dye–based protein stains. Staining with the amine-reactive BODIPY dyes allows the detection of as little as 4 ng of a protein per band in about an hour, with a linear dynamic range of almost two orders of magnitude (Figure 9.64). We offer two colors of BODIPY total-protein blot stains: the green-fluorescent BODIPY FL-X dye, used in combination with DDAO phosphate (Figure 9.63), which produces a red-fluorescent hydrolysis product, and the red-fluorescent BODIPY TR-X dye, used in combination with ELF 39 phosphate (photo, Figure 9.65), which produces a green-fluorescent hydrolysis product. The BODIPY dyes can be visualized using either UV illumination or a laser scanner. The fluorescence signals from the two stains in each kit show very little spectral overlap (Figure 9.66, Figure 9.67) and can be viewed simultaneously and documented separately using the DyeChrome Red/Green Photographic Filter Set (D24771, Accessories for Electrophoresis - Section 23.4). Note that because reaction of the BODIPY succinimidyl ester covalently modifies the protein at random locations, total-protein staining by either of the amine-reactive BODIPY dyes may complicate or preclude subsequent analysis by mass spectrometry or microsequencing.




Figure 9.63 Protein detection with the DyeChrome Western Blot Stain Kit #1 (D21881). Proteins from a rat fibroblast lysate were separated by 2-D gel electrophoresis and blotted onto a PVDF membrane. The proteins are acidic to basic (left to right) and high to low molecular weight (top to bottom). After electrophoresis, the blot was stained with BODIPY FL-X succinimidyl ester (green) to detect total protein. The blot was then incubated with an anti–α-tubulin antibody (A11126), followed by the alkaline phosphatase conjugate of goat anti–mouse IgG antibody, which is included in the kit. Finally, the blot was stained with DDAO phosphate (red). The fluorescent signals were visualized using UV epi-illumination. The signals were documented separately, using the DyeChrome Red/Green Photographic Filter Set (D24771) (A and B), and the resulting images overlaid (C).




Figure 9.64 Linear dynamic range of detection for BODIPY FL-X succinimidyl ester, used as a blot stain. A twofold dilution series of molecular weight markers (P6649) was loaded onto a gel, electrophoresed and electroblotted to a PVDF membrane. The proteins on the blot were then stained with BODIPY FL-X succinimidyl ester, as described for the DyeChrome Western Blot Stain Kits #1, #2 and #3 (D21881, D21882, D21883). The fluorescence intensity for one of the proteins (carbonic anhydrase) was measured and plotted against the amount of protein loaded in the lane. The result shows an approximately linear dynamic range from 4 ng to 125 ng.




Figure 9.66 Fluorescence excitation and emission spectra for the BODIPY FL-X dye and DDAO, products generated in the application of DyeChrome Western Blot Stain Kits #1, #2 and #3 (D21881, D21882, D21883).




Figure 9.67 Fluorescence excitation and emission spectra for the BODIPY TR-X dye and ELF 39 dye, products generated in the application of DyeChrome Western Blot Stain Kits #4, #5 and #6 (D21884, D21885, D21886).


DyeChrome Kits #1, #2 and #3 use the fluorogenic alkaline phosphatase substrate DDAO phosphate, which is rapidly converted to the red-fluorescent product DDAO in the presence of alkaline phosphatase. As little as 1–3 ng of protein per band can be detected with this substrate, depending on the primary antibodies used. To counterstain the entire protein complement on the blot, the kits use BODIPY FL-X succinimidyl ester to stain the proteins with a bright green fluorescence. The fluorescence signals from both stains can be visualized using either UV epi-illumination or visible excitation with a laser scanner.

DyeChrome Kits #1, #2 and #3 include:


DyeChrome Kits #4, #5 and #6 use our proprietary ELF 39 phosphate (Figure 9.65), a novel fluorogenic substrate for alkaline phosphatase that rapidly forms a bright green-fluorescent precipitate at the site of enzyme activity. Sensitive and simple to use, this dye permits the detection of as little as 4–8 ng of a protein per band, depending on the primary antibodies used. The fluorescent signal can be visualized using UV epi-illumination and can be easily separated from that of the red-fluorescent BODIPY TR-X total-protein stain included in the kits as a contrasting stain. BODIPY TR-X staining can be visualized using either UV epi-illumination or visible excitation with a laser scanner. Our DyeChrome Red/Green Photographic Filter Set (D24771, Accessories for Electrophoresis - Section 23.4, Photographic Filters for Fluorescent Dye–Stained Gels and Blots), which contains two specially selected gelatin filters, is recommended for photography of the dichromatic staining using Polaroid 667 black-and-white print film.

DyeChrome Kits #4, #5 and #6 include:


Chemiluminescent Protein Detection on Western Blots

Chemiluminescent enzyme substrates generally provide the most sensitive and background-free method for detecting specific proteins on Western blots. Molecular Probes offers the BOLD APB chemiluminescent substrate (B21901) for detecting alkaline phosphatase conjugates on PVDF or nitrocellulose membranes. Developed at Serologicals Corp., this substrate is based on a 1,2-dioxetane molecule that emits bright chemiluminescence upon reaction with alkaline phosphatase. The BOLD APB chemiluminescent substrate is provided as a 25 mL ready-to-use solution (sufficient for staining 25 minigel blots), making it extremely easy to use (BOLD APB Chemiluminescent Substrate) — there is no need to worry about special blockers or enhancers that are required for other chemiluminescent substrates. The BOLD APB chemiluminescent substrate has several important features:

  • The sensitivity of the BOLD APB substrate is up to 10 times greater than the sensitivity offered by alternative chemiluminescent alkaline phosphatase substrates on PVDF membranes and twofold higher on nitrocellulose membranes.
  • The signal-to-noise ratio of chemiluminescence is exceptionally high, allowing for sensitivity potentially several times that of most fluorescence techniques.
  • The BOLD APB chemiluminescent substrate emits a strong signal that increases in intensity for two hours and remains approximately constant for at least six more hours, allowing plenty of time for the multiple exposures for optimizing detection sensitivity.
  • The BOLD APB chemiluminescent substrate has a five-log dynamic range standard curve.
  • The BOLD APB chemiluminescent substrate is provided as a ready-to-use solution, with a shelf life of at least one year when stored at 4°C.

Although the nature of chemiluminescence precludes the simultaneous detection of multiple colors on the same blot, immunodetection by the BOLD APB chemiluminescent substrate can be paired with sequential staining by the SYPRO Ruby protein blot stain (S11791) for detection of the entire protein profile on the blot (photo). In contrast to fluorescent reagents, chemiluminescent reagents do not require an excitation light source; the energy from a chemical reaction generates light. The chemiluminescent signal can be detected by directly exposing the blot to X-ray film or by using a scanning instrument designed for chemiluminescence.

Chromogenic Protein Detection on Western Blots

Western blotting techniques have conventionally used chromogenic enzyme substrates to detect specific proteins. Substrates for alkaline phosphatase (AP), horseradish peroxidase (HRP) or β-galactosidase have all been used. Conventional chromogenic substrates include:





Figure 4.12 Motor neuron in a three-day chick embryo labeled with lysine-fixable, biotinylated 3000 MW dextran (BDA-3000, D7135). Filled neurons were detected with biotinylated horseradish peroxidase (P917) and diaminobenzidine using standard avidin/streptavidin bridging techniques. Reprinted with permission from ref.

Multiplexed Western Blots: Detecting Multiple Protein Targets Simultaneously

DyeChrome Double Western Blot Stain Kit

The DyeChrome Double Western Blot Stain Kit (D21887) is the first detection kit for multiplexed protein blot staining that permits the use of two different enzyme-conjugated antibodies and a general protein stain for the simultaneous trichromatic detection of multiple targets on the same blot (Figure 9.70). The components of this kit are:

  • A horseradish peroxidase (HRP) conjugate of goat anti–rabbit IgG antibody and the Amplex Gold reagent, for yellow-fluorescent detection of a rabbit antibody to a specific protein or proteins
  • An alkaline phosphatase conjugate of goat anti–mouse IgG antibody and DDAO phosphate, for far-red–fluorescent detection of a mouse antibody to a specific protein or proteins
  • MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone) for blue-fluorescent detection of the total-protein profile
  • Appropriate solvents and buffers for the enzymatic reactions
  • A detailed protocol

Each DyeChrome Double Western Blot Stain Kit contains sufficient materials to stain ~20 minigel blots (6 cm × 9 cm). The two antigens are developed and detected simultaneously; staining is stable indefinitely on dried blots.

Staining with Two Different Labeled Primary Antibodies

To use multiple antibodies on the same blot, the secondary or primary antibodies may also be labeled directly with amine-reactive dyes, such as the succinimidyl esters described in Fluorophores and Their Amine-Reactive Derivatives - Chapter 1. Direct labeling usually provides somewhat lower sensitivity than indirect labeling using enzymatic substrates because the enzymatic substrates can greatly amplify the signal. However, for abundant proteins, direct labeling provides a more streamlined method for staining the blot with multiple antibodies. The limit on the number of colors that can be used together depends only on the compatibility of the antibodies used and the ability of the instrumentation to separate the signals from the fluorescent dyes used (Figure 9.71).




Figure 9.71 Multiplexed detection of two different antibodies on the same blot using fluorophore-labeled secondary antibodies. Cell lysates from nonstimulated (left band) or EGF-stimulated (right band) A431 cells were electrophoresed in an SDS-polyacrylamide gel and blotted. The blot was incubated with primary antibodies against ERK1 and ERK2, p44/42 MAP kinases. Total ERK protein was detected using rabbit anti–ERK IgG antibody followed by anti–rabbit IgG antibody labeled with IRDye 800 (green, Licor). Tyrosine-phosphorylated ERK was detected using mouse anti–phospho-ERK IgG antibody, followed by Alexa Fluor 680 anti–mouse IgG antibody (red, A21057). The blots were imaged using the Odyssey Infrared Imaging System (Licor). Each signal is shown separately (top and middle) and viewed simultaneously on digitally overlaid images (bottom).


As an alternative to directly labeling primary antibodies with an amine-reactive dye, our exceptional Zenon immunolabeling technology (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3) provides an easy, versatile and truly unique method of labeling antibodies with Molecular Probes' premier dyes, haptens and enzymes. This enabling technology not only eliminates the need for secondary detection reagents in many applications, but also simplifies immunolabeling applications that previously were time consuming or impractical, including the use of multiple antibodies derived from the same species in the same protocol, as well as the detection of antibody binding in tissue samples when both the antibody and the tissue are derived from the same species. Moreover, Zenon immunolabeling technology permits the rapid and quantitative preparation of antibody complexes from a purified antibody fraction or from a crude antibody preparation such as serum, ascites fluid or a hybridoma supernatant. Our Zenon reagents may eventually be the only antibody-based detection reagents needed in the laboratory for many high-throughput applications, replacing both direct conjugates of primary antibodies and dye- and enzyme-labeled secondary antibodies in a wide variety of procedures.

Immunoreagents and Labeled Avidins for Use in Western Blot Detection

Fluorescent Avidin Conjugates

Molecular Probes prepares NeutrAvidin biotin-binding protein and streptavidin labeled with a vast assortment of fluorescent dyes (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23), as well as fluorescent microspheres conjugated to streptavidin (Microspheres - Section 6.5). All of these reagents can be used in combination with biotinylated probes for detection of proteins. Although typically not as sensitive as enzyme-amplified techniques, fluorescent avidin conjugates are easy to use and permit multicolor detection of targets.

Primary Antibodies

Western blotting relies on immunostaining with antibodies to specific proteins. Molecular Probes has available a variety of primary antibodies that are useful for detecting specific proteins on blotting membranes. These include antibodies directed against:


Molecular Probes also provides antibodies against epitope and protein tags for detecting appropriately tagged recombinant proteins:





Figure 9.72 Detection of HA-fusion proteins using anti-HA antibody. Six proteins, each fused to the HA domain, were electrophoresed through a 13% polyacrylamide gel and blotted onto a PVDF membrane. The blot was incubated with the Alexa Fluor 488 conjugate of anti-HA antibody (A21287), followed by rabbit anti–Alexa Fluor 488 antibody (A11094). The antibody complex was then detected using the Amplex Gold Western Blot Stain Kit #2 (A21891).


Finally, our anti-dye, anti-biotin, anti-DNP and anti-nitrotyrosine (Figure 18.23) antibodies can be employed for the selective detection of primary or secondary proteins labeled with fluorescent dyes, biotin, DSB-X biotin or the DNP or o-nitrophenol haptens (Anti-Dye and Anti-Hapten Antibodies - Section 7.4, Anti-fluorophore antibodies and their conjugates - Table 7.19). Biotinylation and Haptenylation Reagents - Section 4.2 describes our recommended reagents for labeling proteins and nucleic acids with biotin (Biotinylation and desthiobiotinylation reagents - Table 4.1) and haptens (Selected haptenylation reagents and their anti-hapten antibodies - Table 4.2).

Fluorescence-Based Detection of Oligohistidine Fusion Proteins

The oligohistidine domain is a Ni2+-binding peptide sequence comprising a string of four to six histidine residues. When the DNA sequence corresponding to the oligohistidine domain is fused in frame with a gene of interest, the resulting recombinant protein can be easily purified using a nickel-chelating resin.ref Molecular Probes has developed technologies that make it possible to quickly and easily identify oligohistidine fusion proteins in gels or on blots.

Pro-Q Sapphire 532 Oligohistidine Gel Stain

Traditional analysis of fusion proteins containing an oligohistidine domain has generally required purification using a nickel-chelating resin followed by protein separation with SDS-polyacrylamide gel electrophoresis and Western blot analysis. With the Pro-Q Sapphire 532 oligohistidine gel stain (P33354), oligohistidine fusion proteins can be detected directly in an SDS-polyacrylamide gel (photo), eliminating the need to blot the protein to a membrane. The Pro-Q Sapphire 532 oligohistidine gel stain consists of a proprietary fluorescent dye selective for oligohistidine domains. Staining is complete within hours, and as little as 15 ng of a hexahistidine fusion protein can be detected. The fluorescence intensity of the stained protein varies somewhat with the fusion protein, indicating that the dye binding may be dependent on protein context. Typically, a band containing 30–50 ng of oligohistidine fusion protein can be detected in a minigel. Samples run in a standard-size gel may be more difficult to detect because the protein is more dispersed in the larger well and thicker gel. Note that with highly basic proteins, weak crossreactivity with the reagent may occur. For weakly expressed proteins requiring higher sensitivity, the Pro-Q Oligohistidine Blot Stain Kits (P21878, P21879) are recommended.

Pro-Q Sapphire 532 stain has excitation/emission maxima of approximately 535/572 nm, so it is optimally excited with 532 nm laser scanners. However, because the excitation spectrum is broad, the stain can also be detected after excitation by 473 nm or 488 nm blue lasers or by UV illumination. After documenting the oligohistidine signal, the gel can be stained for total protein using SYPRO Ruby protein gel stain.

Pro-Q Sapphire 365 and Pro-Q Sapphire 488 Oligohistidine Gel Stains

The Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine gel stains (P21876, P21877) provide a simple method for detecting oligohistidine fusion proteins directly in an SDS-polyacrylamide gel (photo, photo), eliminating the need to blot the protein to a membrane.ref These proprietary reagents each comprise a state-of-the-art fluorescent dye conjugated to a nitrilotriacetic acid (NTA) moiety. The staining procedure is very simple — simply fix the gel and incubate it with the stain. The NTA moiety chelates Ni2+ bound by the oligohistidine domain, resulting in optimal staining in just 45 minutes. Note that because the NTA is negatively charged, there may also be some weak crossreactivity with highly basic proteins. The Pro-Q Sapphire 365 oligohistidine gel stain (P21876, Pro-Q(R) Sapphire 365 Oligohistidine Gel Stain) can be viewed using 365 nm UV illumination and the SYPRO photographic filter (S6656, Accessories for Electrophoresis - Section 23.4, Figure 23.51); it has a sensitivity limit of ~30 ng/band of an oligohistidine fusion protein. The Pro-Q Sapphire 488 oligohistidine gel stain (P21877, Pro-Q(R) Sapphire 488 Oligohistidine Gel Stain) can be viewed using visible light with wavelengths near its 510 nm excitation maximum and has a sensitivity limit of ~30 ng/band. These limits of sensitivity were determined using a hexahistidine–urate oxidase fusion protein; other fusion proteins we have tested show levels of sensitivity between 60 and 100 ng per band, suggesting that the protein environment may have an effect on the ability of NTA-based compounds to bind to oligohistidine domains. After documenting the oligohistidine signal, the total-protein profile can be visualized using the SYPRO Ruby protein gel stain (Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3, photo).

Pro-Q Oligohistidine Blot Stain Kits

The Pro-Q Oligohistidine Blot Stain Kits provide a simple, fast and sensitive method for the detection of oligohistidine fusion proteins on PVDF membranes. The staining technique uses biotin-X nitrilotriacetic acid (biotin-X NTA, structure), which chelates Ni2+. The blot is incubated with a complex of biotin-X NTA, Ni2+ and alkaline phosphatase streptavidin. Within 20 minutes, the complex binds to oligohistidine fusion proteins. The complex is then detected using a fluorogenic alkaline phosphatase substrate — either DDAO phosphate (in Kit #1, P21878; photo; Pro-Q(R) Oligohistidine Blot Stain Kit #1 with biotin NTA, streptavidin and DDAO phosphate), which produces a red-fluorescent product, or ELF 39 phosphate (in Kit #2, P21879; Pro-Q(R) Oligohistidine Blot Stain Kit #2; Figure 9.65), which produces a green-fluorescent product. Both substrates (described in Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3 and above under the heading "Western Blot Stain Kits and Reagents" provide very rapid and sensitive detection of the alkaline phosphatase conjugates, making it possible to detect as little as ~16 ng/band in less than 90 minutes after blotting, depending on the particular fusion protein. The sensitivity of these fluorogenic substrates rivals that of chemiluminescence detection. However, because the fluorescent products are chemically stable, there is no need to perform the reaction in a darkroom or to incubate the blot with X-ray film. Furthermore, the fluorescent signal, unlike transient chemiluminescent signals, can be imaged several times and is stable indefinitely on dried blots. The biotin-X NTA and DDAO can be removed from the blot for restaining with another detection method; the ELF 39 stain, however, is permanent. Biotin-X NTA is also available separately (B11790).

Penta·His Antibody

Developed by QIAGEN, the Penta·His mouse IgG1 monoclonal antibody (P21315, Penta-His Mouse Monoclonal Antibody (Anti-Pentahistidine)) provides a sensitive method for specific detection of fusion proteins that have an oligohistidine domain comprising five or six consecutive histidine residues. The antibody does not recognize tetrahistidine domains or domains in which the histidine string is interrupted by another amino acid. The Penta·His antibody binds to the oligohistidine domain regardless of the surrounding amino acid context and even when the group is partially hidden, although subtle differences in the amino acid context may change the sensitivity limit for a particular fusion protein. The antibody is ideal for detecting oligohistidine fusion proteins on blots in combination with our Western Blot Stain Kits (photo; see the description of our Amplex Gold, Pro-Q and DyeChrome Western Blot Stain Kits above). The Penta·His antibody is also useful for immunoprecipitation, ELISA assays, and immunohistochemistry.

Other Specialized Techniques for Detecting Specific Proteins in Gels and on Blots

Detecting Calcium-Binding Proteins in Gels

The luminescent lanthanide terbium, which is available from Molecular Probes as its chloride salt (Tb3+ from TbCl3, T1247), selectively stains calcium-binding proteins in SDS-polyacrylamide gels.ref With some modifications to the staining protocol, these lanthanides can also be used to detect all protein bands.ref Terbium chloride has also been used as a rapid negative stain for proteins in SDS-polyacrylamide gels, in which the background is green fluorescent and the proteins are unstained.ref

Detecting Glutathiolation

Biotinylated glutathione ethyl ester (BioGEE, G36000) is a cell-permeant, biotinylated glutathione analog for detecting glutathiolation. Under conditions of oxidative stress, cells may transiently incorporate glutathione into proteins. Stressed cells incubated with BioGEE will also incorporate this biotinylated glutathione derivative into proteins, facilitating the identification of oxidation-sensitive proteins.ref Once these cells are fixed and permeabilized, glutathiolation levels can be detected with a fluorescent streptavidin conjugate (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23) using either flow cytometry or fluorescence microscopy. Proteins glutathiolated with BioGEE can also be extracted and analyzed by mass spectrometry or by Western blotting methods in conjunction with fluorophore- or enzyme-labeled streptavidin conjugates.

Detecting Penicillin-Binding Proteins

BOCILLIN FL penicillin and BOCILLIN 650/665 penicillin (B13233, B13234) are green- and infrared-fluorescent penicillin analogs, respectively, that bind selectively and with high affinity to penicillin-binding proteins present on the cytoplasmic membranes of eubacteria.ref When electrophoresed under nonreducing conditions, the dye-labeled penicillin-binding proteins are easily visible in the gel with sensitivity in the low nanogram range ref (Figure 9.78). BOCILLIN FL penicillin, synthesized from penicillin V and the BODIPY FL dye (spectrally similar to fluorescein), has been used to determine the penicillin-binding protein profiles of Escherichia coli, Pseudomonas aeruginosa and Streptococcus pneumoniae, and these binding profiles are found to be similar to those reported by researchers using radioactively labeled penicillin V.ref Fluorescently labeled penicillin has also been used for direct labeling and rapid detection of whole E. coli and Bacillus licheniformis ref and of Enterobacter pneumoniae.ref The β-lactam sensor-transducer (BlaR), an integral membrane protein from Staphylococcus aureus, covalently and stoichiometrically reacts with β-lactam antibiotics, including BOCILLIN FL penicillin, by acylation of its active-site serine residue.ref




Figure 9.78 Detection of penicillin-binding proteins (PBPs) from Escherichia coli and Pseudomonas aeruginosa.ref The membrane fractions from E. coli and P. aeruginosa were prepared as previously described ref and labeled with BOCILLIN 650/665 penicillin (B13234). The labeled membranes were separated on an SDS-polyacrylamide gel, stained with SYPRO Ruby and visualized using a Typhoon imager (Molecular Dynamics). The location of PBPs from E. coli are labeled to the left of the gels. Lanes 1, 3, and 5 are E. coli membrane preparations; lanes 2, 4, and 6 are P. aeruginosa membrane preparations; lanes 1 and 2 are overlays of images obtained from total protein (green) and PBP (red) scans; lanes 3 and 4 are total protein visualized with the SYPRO Ruby protein gel stain; lanes 5 and 6 are PBPs as detected by BOCILLIN 650/665. Image used with permission from Wiley VCH publishers.


Detecting Enzymes in Nondenaturing Gels

Many different enzymes have been detected in nondenaturing gels by using various chromogenic substrates, including X-Gal (B1690, B22015; Detecting Glycosidases - Section 10.2), X-GlcU (B1691, Detecting Glycosidases - Section 10.2) and NBT/BCIP ref (N6495, B6492; Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3). In unpublished experiments, we have shown that our ELF 97 phosphatase substrate (E6589, Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3) forms a highly fluorescent precipitate at the site of enzymatic activity (either acid or alkaline phosphatase activity) in nondenaturing polyacrylamide gels. In addition, we have demonstrated that our ELF 97 β-D-glucuronidase substrate (E6587, Detecting Glycosidases - Section 10.2) has similar utility for detecting β-glucuronidase in native or SDS-polyacrylamide gels, with a detection limit of less than 5 ng of the enzyme ref (Figure 10.24). The ELF 97 β-D-glucuronidase substrate can also be used in combination with our SYPRO Tangerine protein gel stain (S12010, Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3) for detecting the total-protein profile in the gel (Figure 9.29). Fluorogenic protease substrates based on the rhodamine 110 dye (Detecting Peptidases and Proteases - Section 10.4, Rhodamine 110-based bis-peptide substrates - Table 10.2) have been applied by overlaying filter paper impregnated with the substrates on SDS-polyacrylamide gels to detect protease activity.ref

Detecting Protein Functional Groups in Gels and on Blots

Several of the low molecular weight, thiol-reactive reagents described in Thiol-Reactive Probes - Chapter 2 can potentially be used to selectively detect thiol-containing proteins in gels without appreciable staining of proteins that do not contain thiols. Compounds that may be particularly useful include BODIPY 493/503 methyl bromide and BODIPY 630/650 methyl bromide (B2103, B22802; Thiol-Reactive Probes Excited with Visible Light - Section 2.2), IANBD amide (D2004, Thiol-Reactive Probes Excited with Visible Light - Section 2.2), monochlorobimane and monobromobimane ref (M1381MP, M1378; FluoroPure Grade - Note 19.2, M20381; Thiol-Reactive Probes Excited with Ultraviolet Light - Section 2.3), the coumarin iodoacetamide IDCC (D20382, Thiol-Reactive Probes Excited with Ultraviolet Light - Section 2.3) and CellTracker Blue CMAC (C2110, Membrane-Permeant Reactive Tracers - Section 14.2). These selected compounds are all electrically neutral reagents and thus do not appreciably change the charge or mass of the protein, a feature that may make them useful for derivatizing the thiolated protein prior to separation by isoelectric focusing. Monobromobimane has been used to derivatize thiol-containing proteins prior to separation by isoelectric focusing without the modification having an appreciable effect on the protein's electrophoretic mobility.ref 8-Aminonaphthalene-1,3,6-trisulfonic acid (A350, Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2) has been used to directly stain periodate-oxidized glycoproteins on PVDF membranes.ref Glycoprotein binding to PVDF membranes was selectively enhanced by pre-coating the membrane with wheat germ agglutinin (WGA). Alexa Fluor 350 hydrazide (A10439, Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2) has been used similarly for glycoprotein detection both in gels and on blots.ref

Chemical Labeling of Nascent Proteins

The relatively compact BODIPY FL fluorophore (D6140, BODIPY Dye Series - Section 1.4) has been used as a fluorescent reporter group in nascent proteins. This dye was incorporated at the N-terminus of nascent proteins using an Escherichia coli tRNA(fmet) misaminoacylated with a methionine containing a BODIPY FL fluorophore at its amino group.ref Under optimal conditions, subnanogram quantities of green-fluorescent bands from in vitro–produced fluorescent proteins can be detected by gel electrophoresis using a laser scanner.

Mitochondrial Protein Extracts

For researchers seeking a source of mitochondrial protein standards, Molecular Probes offers human heart mitochondrial proteins for SDS-polyacrylamide gel electrophoresis (M22430). This complete mitochondrial lysate has tested negative for hepatitis B and C, as well as HIV 1 and 2 in serology tests. Mitochondrial protein extracts are useful for comparing new mitochondrial protein preparations in SDS-polyacrylamide gels and for testing mitochondrial antibodies.

Rhinohide Polyacrylamide Gel Strengthener

Our Rhinohide polyacrylamide gel strengthener improves upon classic polyacrylamide gel technology by making gels much stronger, providing easier handling and much greater resistance to tearing without adverse side effects (Figure 9.34). Rhinohide polyacrylamide gel strengthener is highly recommended for low-percentage gels, large-format gels and gels subject to multiple staining and handling steps.

SDS-polyacrylamide gels supplemented with Rhinohide polyacrylamide gel strengthener exhibit resolution capabilities comparable to traditional SDS-polyacrylamide gels, with clear, focused bands and without the undesirable side effects common for other gel strengtheners.ref For example, film-backed gels and polyester fabric–reinforced gels interfere with blotting techniques and can negatively affect protein staining. Alternatively, strengthening gels by the addition of pre-formed polymers causes turbidity and can produce serious spot-morphology artifacts, such as the distortion of high molecular weight bands or doubling of protein spots in the molecular weight dimension of 2-D gels.ref

Rhinohide polyacrylamide gel strengthener produces gels with excellent transparency, providing exceptional image viewing and scanning of fluorescently stained gels with minimal background staining. Compatible with silver and Coomassie staining, it is also the perfect companion to our Multiplexed Proteomics technology described earlier in this section. Our Rhinohide Polyacrylamide Gel Strengthener Kit (R33410) includes:


This kit provides sufficient materials for making 1 L of a 30% acrylamide/bis-acrylamide stock solution containing the Rhinohide gel strengthener. We also offer a concentrated form of the Rhinohide polyacrylamide gel strengthener (R33400) for adding to existing stock solutions of acrylamide/bis-acrylamide (37.5:1), as well as the acrylamide/bis-acrylamide mixture (A33405) for making these stock solutions. Because prestained proteins, such as prestained molecular weight markers, will not migrate correctly in acrylamide gels containing the Rhinohide polyacrylamide gel strengthener, we recommend using only unstained proteins as markers.