Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3

The luminescent SYPRO protein gel stains are revolutionizing the detection of the total-protein profile in polyacrylamide gels. These novel protein detection reagents combine several characteristics that together make them far superior to traditional staining methods, including:

• High sensitivity, making it possible to detect even minimally expressed proteins
• Fast and easy staining protocols, simplifying the processing of multiple gels or blots
• Minimal protein-to-protein variation in staining, allowing quantitative comparisons between proteins
• Broad linear quantitation range, an essential property for performing comparative protein expression studies
• Compatibility with subsequent microanalysis, streamlining techniques such as immunostaining, microsequencing and mass spectrometry
• No nucleic acid staining or polysaccharide staining, allowing analysis of relatively impure or contaminated samples
• Instrument compatibility, making the dyes suitable for research labs with either simple UV transilluminators or laser scanners

Currently the most common methods for universal profiling of proteins in gels are Coomassie brilliant blue staining and silver staining. Although Coomassie brilliant blue is an inexpensive reagent, its staining is relatively insensitive and, because it requires destaining, time consuming. Silver staining may be up to 100 times more sensitive than Coomassie brilliant blue staining, but it is relatively expensive and entails several labor-intensive and time-sensitive steps. Silver staining also exhibits a high degree of protein-to-protein variability; staining intensity and color are very dependent on each polypeptide's sequence and degree of glycosylation, and some proteins are detectable only as negatively stained patches. Moreover, silver staining shows very poor linearity with protein concentration (Figure 9.17) and poor reproducibility in staining from gel to gel, making it inadequate for comparative studies of protein expression in cells. The drawbacks of these traditional stains can all be overcome by using one of the SYPRO stains, without sacrificing detection sensitivity. We have developed a SYPRO dye optimized for protein profiling in nearly every type of gel (Summary of SYPRO and Coomassie Fluor luminescent and fluorescent protein gel stains - Table 9.3) or blot application (Summary of fluorescent and luminescent protein blot stains - Table 9.4). The characteristics and applications of the individual SYPRO protein gel and blot stains for detecting the total-protein profile of a sample are described in this section. Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4 discusses combining these SYPRO stains with selective protein detection reagents for multiparameter staining with our Multiplexed Proteomics technology.

Figure 9.17 Quantitation of proteins in gels using SYPRO Ruby protein gel stain versus silver stain. Dilutions of proteins were electrophoresed on eight different SDS-polyacrylamide gels, two gels for each of four dilution ranges. The gels were stained with either SYPRO Ruby protein gel stain (S12000, S12001, S21900) or a silver stain. Staining intensities were quantitated using either the Fluor-S MAX MultiImager documentation system (Bio-Rad Laboratories) or the FLA3000G laser scanner (Fuji Photo Film Co.) and plotted against the protein amount for bovine serum albumin. SYPRO Ruby protein gel stain shows a linear quantitation range over three orders of magnitude, as well as consistent staining intensities from gel to gel. In contrast, the silver stain shows linear quantitation over only a small range, a very shallow slope and inconsistent staining intensities from gel to gel, even when corrected for background differences.

SYPRO Ruby Protein Gel Stain: Ultrasensitive Protein Detection in 1-D, 2-D and IEF Gels

Our Patented SYPRO Ruby protein gel stain (S12000, S12001, S21900; SYPRO(R) Ruby Protein Gel Stain) is a ready-to-use protein stain that has sensitivity equal to or exceeding that of the best silver staining techniques, is compatible with Edman sequencing and mass spectrometry and can be visualized with a simple UV transilluminator or a laser scanner. SYPRO Ruby protein gel stain has characteristics that make it far superior to conventional staining techniques:

• High-sensitivity staining. SYPRO Ruby protein gel stain provides at least the same subnanogram sensitivity as the best silver staining techniques in 1-D, 2-D or IEF gels (Figure 9.18).
• Simple protocol. After fixation, the gel is incubated in the staining solution (Figure 9.19). No stop solutions or destaining steps are required and, unlike silver staining, gels can be left in the dye solution for indefinite periods without overstaining, vastly simplifying the simultaneous processing of multiple gels and making it possible to perform high-throughput staining without investing in robotic staining devices (SYPRO(R) Ruby Protein Gel Stain).
• Accurate peptide and protein detection. SYPRO Ruby protein gel stain shows little protein-to-protein variability in staining and detects some proteins that are completely missed by silver staining (Figure 9.20), such as heavily glycosylated proteins. Unlike silver staining, SYPRO Ruby dye does not stain extraneous nucleic acids, lipids or carbohydrates in the sample.
• Excellent performance in comparative protein expression studies. SYPRO Ruby stain shows a greater linear quantitation range than either silver or Coomassie brilliant blue staining — extending over three orders of magnitude — making it possible to accurately compare protein expression levels (Figure 9.17, Figure 9.18). Gel-to-gel staining is extremely consistent; same-spot intensity comparisons between identical 2-D gels show a correlation coefficient of 0.9. Multiple gels can easily be compared using available software (). No other protein quantitation method, including running multiple prestained samples on the same gel, gives results that approach this level of discrimination.
• Compatibility with microsequencing and mass spectrometry. Unlike silver staining techniques, which use glutaraldehyde- or formaldehyde-based fixatives, SYPRO Ruby dye is a gentle stain that interacts noncovalently with proteins. Thus, high-quality Edman sequencing or mass spectrometry data (Figure 9.22) can be obtained immediately after staining, without modification steps that may compromise sensitivity. Automated in-gel digestion methods have been used in the analysis of femtomole levels of SYPRO Ruby dye–stained proteins.
• Utility for isoelectric focusing (IEF). SYPRO Ruby protein gel stain also provides reliable, high-sensitivity staining for isoelectric focusing (IEF) gels (Figure 9.23) without the problems typically encountered with silver staining, such as ampholyte staining or mirroring effects on the plastic gel backing.
• Easily visualized signal. SYPRO Ruby protein gel stain comprises the transition metal ruthenium, which shows an extremely bright and photostable red-orange luminescence when excited with either UV or blue light (Figure 9.24). Stained proteins can be visualized using a UV transilluminator, a blue-light transilluminator or a laser-scanning instrument. Gels can then be documented using Polaroid 667 black-and-white print film, a CCD camera with an image documentation system or a laser-scanning instrument (Imaging platforms validated as suitable for visualization of SYPRO Ruby protein stains - Table 9.5). For optimal sensitivity using a UV transilluminator and Polaroid 667 black-and-white print film, the SYPRO photographic filter (S6656, Figure 23.51) is recommended.
• Minimal hazardous waste. The amount of hazardous waste generated with the SYPRO Ruby protein gel stain is greatly reduced as compared with that of silver staining, minimizing the hassles and expense associated with waste disposal.

Figure 9.18 Amounts of carbonic anhydrase ranging from 1 ng to 1000 ng were separated on an SDS-polyacrylamide gel and stained with the SYPRO Ruby protein gel stain (S12000, S12001, S21900). The inset shows the excellent linearity in the lower part of the range from 1 ng to 60 ng protein. Staining intensities were quantitated using the Molecular Imager FX documentation system (Bio-Rad Laboratories). For comparison, the gray band shows the linear range for the same protein detected with silver staining.




Figure 9.19 Staining gels with SYPRO Ruby protein gel stain (S12000, S12001, S21900) is simple: just fix, stain and wash.




Figure 9.20 SYPRO Ruby protein gel stain (S12000, S12001, S21900) shows less protein-to-protein variation than silver staining. Proteins from a cell lysate were run on a 2-D gel and stained with SYPRO Ruby protein gel stain (left) or silver stain (right). The grayscale values of the SYPRO Ruby dye–stained gel have been inverted for easier comparison with the silver-stained gel.




Figure 9.22 Mass spectrum profile of NADH:ubiquinone reductase precursor (75,000-dalton subunit) obtained after 2-D gel electrophoresis of bovine heart mitochondria and staining with SYPRO Ruby protein gel stain (S12000, S12001, S21900). Bovine heart mitochondria were a gift of Roderick Capaldi, University of Oregon.





Figure 9.23 SYPRO Ruby protein gel stain versus silver stain for IEF gels. Serial dilutions of isoelectric focusing protein standards were electrophoresed on two identical polyacrylamide gels. One gel was stained with SYPRO Ruby protein gel stain (S12000, S12001, S21900) (left) and the other with silver stain (right). Both stains show a similar limit of sensitivity for all proteins.





Figure 9.24 Luminescence excitation (dashed line) and emission (solid line) spectra of the SYPRO Ruby protein gel and blot stains (S11791, S12000, S12001, S21900).




Figure 23.51 Transmittance profile of the SYBR Safe photographic filter (S37100) and the SYPRO photographic filter (S6656), which are identical.

SYPRO Ruby protein gel stain is supplied as 200 mL of a 1X staining solution (S12001), sufficient for staining about four minigels, or 1 L of a 1X staining solution (S12000), sufficient for staining about 20 minigels or two standard 2-D gels. Additionally, we offer SYPRO Ruby protein gel stain in a 5 L box (S21900), sufficient for staining about 100 minigels or 10 standard 2-D gels. These boxes are easy to stack and store, and the convenient spigot makes it easy to dispense just the right amount of stain (Figure 9.25). Significant discounts are available for multiple-unit purchases of the SYPRO Ruby products. All of the SYPRO Ruby protein gel stains are accompanied by detailed instructions for staining and photography of gels (SYPRO(R) Ruby Protein Gel Stain).

Figure 9.25 SYPRO Ruby protein gel stain is available in 200 mL, 1 L or 5 L sizes (S12000, S12001, S21900). Generous bulk discounts are available for multiple-unit purchases of these products.

SYPRO Orange and SYPRO Red Protein Gel Stains: For Routine Detection of Proteins in 1-D SDS-Polyacrylamide Gels

Molecular Probes' Patented SYPRO Orange (S6650, S6651) and SYPRO Red (S6653, S6654) protein gel stains provide a fluorescence-based alternative for protein detection in SDS-polyacrylamide gels that is not only faster and more sensitive than Coomassie brilliant blue staining, but can be as sensitive as short-protocol silver staining methods (Figure 9.26) at a fraction of the time, effort and cost of silver staining (SYPRO(R) Orange and SYPRO(R) Red Protein Gel Stains). In the presence of excess SDS, nonpolar regions of polypeptides are coated with detergent molecules, forming a micelle-like structure with a nearly constant SDS/protein ratio (1.4 g SDS:1.0 g protein); this constant charge-per-mass ratio is the basis of molecular weight determination by SDS-polyacrylamide gel electrophoresis. The SYPRO Orange and SYPRO Red dyes appear to bind to the SDS coat that surrounds proteins in SDS-polyacrylamide gels. Thus, the staining observed with these dyes exhibits relatively little protein-to-protein variation and is linearly related to protein mass (Figure 9.27). Some important features of the SYPRO Orange and SYPRO Red protein gel stains include:

• Ease of use. Following electrophoresis, the gel is stained for 10–60 minutes and then briefly rinsed — no separate fixation, stop or destaining steps are required. After staining, the gel is immediately ready for photography, or it can be stored, in or out of the staining solution, for days (SYPRO(R) Orange and SYPRO(R) Red Protein Gel Stains).
• High sensitivity. The SYPRO Orange and SYPRO Red protein gel stains routinely provide a sensitivity level of at least 8–16 ng per band in SDS-polyacrylamide minigels when visualized with standard 300 nm transillumination (Figure 9.26). Photography using Polaroid 667 black-and-white print film and a SYPRO photographic filter (S6656, Photographic Filters for Fluorescent Dye–Stained Gels and Blots, Figure 23.51) enhances the detection of staining with SYPRO Orange or SYPRO Red dye by several-fold over visible detection because the film integrates the signal throughout the duration of the exposure. Laser scanners also detect nanogram quantities of SYPRO dye–stained proteins in gels.
• Broad linear quantitation range. Protein detection in gels stained with either the SYPRO Orange or SYPRO Red stain is linear over three orders of magnitude in protein quantity (Figure 9.27).
• Uniform protein staining. Unlike silver staining, the SYPRO Orange and SYPRO Red dyes exhibit relatively low protein-to-protein variability in SDS-polyacrylamide gels (Figure 9.27) and do not stain nucleic acids, which are sometimes found in protein mixtures from cell or tissue extracts. In addition, the SYPRO Orange and SYPRO Red dyes only weakly stain lipopolysaccharides in bacterial lysates, whereas these biopolymers are strongly detected by some types of silver staining. Glycoproteins (such as the IgG variable subunit) and proteins with prosthetic groups (such as bovine cytochrome oxidase) are also efficiently stained with the SYPRO Orange and SYPRO Red dyes.
• Photostability. Proteins stained with the SYPRO Orange or SYPRO Red dye are relatively photostable, enabling the researcher to acquire multiple photographic images and to use long film-exposure times (2–8 seconds). Gels that are illuminated for long periods of time may partially photobleach but can be restained with little loss of sensitivity.
• Compatibility with many types of instruments. Although their excitation maxima are in the visible range (Figure 9.28), SYPRO dye–stained gels are readily visualized using standard 300 nm transilluminators. SYPRO Orange protein gel stain also exhibits good sensitivity when viewed with a blue-light transilluminator, and both SYPRO Orange and SYPRO Red protein gel stains are suitable for use with many laser-scanning instruments.
• Chemical stability. The SYPRO Orange and SYPRO Red gel stains are chemically stable; if protected from light, fluorescence of the stained gel is stable for several days, and staining solutions can be stored for months.
• Economy. The SYPRO Orange and SYPRO Red gel stains are not only less expensive than silver-staining kits but faster and less laborious to use. Additionally, use of the SYPRO Orange or SYPRO Red dye avoids the costs of purchase and disposal of large amounts of organic solvents that are required for Coomassie brilliant blue staining. Significant discounts are available on multiple-unit purchases of all of the SYPRO dyes for high-throughput research applications.
• Compatibility with mass spectroscopy. Unlike silver staining, the SYPRO Orange and SYPRO Red dyes do not covalently bind to proteins, allowing subsequent analysis of stained proteins by microsequencing or mass spectrometry.

Figure 9.26 Comparison of the sensitivity achieved with SYPRO Orange, SYPRO Red, silver and Coomassie brilliant blue stains. Identical SDS-polyacrylamide gels were stained with A) SYPRO Orange protein gel stain (S6650, S6651), B) SYPRO Red protein gel stain (S6653, S6654), C) silver stain and D) Coomassie brilliant blue stain, according to standard protocols. The SYPRO Orange and SYPRO Red dye–stained gels were photographed using 300 nm transillumination, a SYPRO photographic filter (S6656) and Polaroid 667 black-and-white print film. The silver- and Coomassie brilliant blue–stained gels were photographed with transmitted white light and Polaroid 667 black-and-white print film; no photographic filter was used to photograph these gels.




Figure 9.27 Quantitation of proteins in a gel using SYPRO Orange protein gel stain (S6650, S6651). A protein mixture was serially diluted and electrophoresed on a 15% SDS-polyacrylamide gel and then stained with SYPRO Orange protein gel stain. The gel was then scanned using a Molecular Dynamics Storm gel and blot analysis system (excitation/emission 488/>520 nm) and analyzed to yield the fluorescence intensities of the stained bands. The fluorescence intensity scale is the same in both panels, illustrating the minimal protein-to-protein staining variation of the SYPRO Orange gel stain. Detection limits are between 2 and 16 ng of protein; the linear detection ranges are approximately 1000-fold. Proteins represented are: A) β-galactosidase (), lysozyme (), bovine serum albumin (BSA, ) and phosphorylase B (); B) myosin (), soybean trypsin inhibitor (), ovalbumin () and carbonic anhydrase ().




Figure 9.28 Luminescence excitation and emission spectra of A) SYPRO Orange (S6650, S6651) and B) SYPRO Red (S6653, S6654) protein gel stains diluted 1:10,000 in water containing 0.05% SDS and 150 µg/mL bovine serum albumin (BSA).

The SYPRO Orange and SYPRO Red stains have very similar staining properties, though we have observed that proteins stained with the SYPRO Orange dye are slightly brighter, whereas gels stained with the SYPRO Red dye tend to have lower background fluorescence. For maximum sensitivity with UV transilluminators, we recommend documenting the signal using Polaroid 667 black-and-white print film and the SYPRO photographic filter (S6656, Figure 23.51, Photographic Filters for Fluorescent Dye–Stained Gels and Blots). For maximum sensitivity with laser scanners, we recommend matching the appropriate SYPRO dye with the excitation light source of the instrument. For instance, SYPRO Orange protein gel stain is most suitable for gel scanners that employ argon-ion lasers with output at 488 nm, whereas SYPRO Red protein gel stain is best matched to laser-scanning instruments that employ Nd:YAG lasers with output at 532 nm. Interestingly, the SYPRO Red dye is also compatible with scanners using excitation by the 633 nm spectral line of the He–Ne laser. The SYPRO Red protein gel stain has been used as a prestain for protein analysis in an automated ultrathin-layer gel electrophoretic technique. The SYPRO Orange protein gel stain has been used for protein sizing on a microchip and for analyzing the kinetics of isothermal protein denaturation.

The SYPRO Orange and SYPRO Red protein gel stains are compatible with SDS or urea/SDS gels. Staining native proteins in gels in the absence of SDS results in more protein-to-protein variation and lower sensitivity than staining SDS-denatured proteins, due to variations in hydrophobicity of the target polypeptides. However, sensitivity of SYPRO dye staining in native gels can be improved if gels are soaked in 0.05% SDS solution after electrophoresis but prior to staining. For optimal staining of proteins in 2-D gels and IEF gels, we recommend the SYPRO Ruby protein gel stain (S12000, S12001, S21900; see above).

Because the SYPRO Orange and SYPRO Red dyes do not covalently bind to proteins, stained proteins can be subsequently analyzed by microsequencing or mass spectrometry. However, these dyes are not recommended for staining gels prior to blotting, as there is a significant loss of sensitivity when proteins are stained with the SYPRO Orange or SYPRO Red dyes in typical Western blotting buffers. For maximum sensitivity and ease of use in staining proteins on blots, we recommend the use of SYPRO Tangerine protein gel stain (S12010, see below) to stain proteins on the gel before blotting, or SYPRO Ruby or SYPRO Rose Plus protein blot stains (S11791, S12011; see below) for staining proteins on nitrocellulose or PVDF membranes after blotting.

The Patented SYPRO Orange and SYPRO Red protein gel stains are available as 500 µL stock solutions in dimethylsulfoxide (DMSO), either in a single vial (S6650, S6653) or specially packaged as a set of 10 vials, each containing 50 µL (S6651, S6654; SYPRO(R) Orange and SYPRO(R) Red Protein Gel Stains). The reagents are supplied as 5000X concentrates; thus, 500 µL of either stain yields 2.5 L of staining solution. Significant discounts are available on multiple-unit purchases of all of the SYPRO dyes for high-throughput research applications. Photography of proteins in gels, which is essential for obtaining the maximum sensitivity, requires use of the SYPRO photographic filter (S6656, Photographic Filters for Fluorescent Dye–Stained Gels and Blots).

SYPRO Tangerine Protein Gel Stain: Sensitive Protein Staining without Fixation for Electroelution, Zymography and Classroom Use

Our SYPRO Tangerine protein gel stain (S12010, SYPRO(R) Tangerine Protein Gel Stain) stains proteins in gels without the need for either acids or organic solvents and thus serves as an alternative to conventional protein stains that fix proteins in the gel. Whereas the SYPRO Orange and SYPRO Red stains require a staining solution containing acetic acid for maximum performance, staining with the SYPRO Tangerine protein gel stain is possible in almost any buffer that contains NaCl. Because proteins are not fixed during the staining procedure, they can be readily eluted from gels, used for zymography (in-gel enzyme activity assays, Figure 9.29) or analyzed by mass spectrometry. The SYPRO Tangerine stain can also be used to stain gels before transferring the proteins to nitrocellulose or PVDF membranes for immunostaining (Western blotting). Like the SYPRO Orange and SYPRO Red protein gel stains, the SYPRO Tangerine protein gel stain shows high sensitivity (down to ~4 ng/band) and a broad linear quantitation range (Figure 9.30). Environmentally friendly and easy to use, the SYPRO Tangerine protein gel stain is also ideal for use in educational settings, especially when used with UV-free blue-light transilluminators.

The SYPRO Tangerine stain is compatible with conventional SDS-polyacrylamide gel electrophoresis, but it is not recommended for 2-D or IEF gels. Stained proteins can be visualized using a UV transilluminator, a blue-light transilluminator or a laser scanner. Photography of stained gels using Polaroid 667 black-and-white print film requires use of the SYPRO photographic filter (S6656, Photographic Filters for Fluorescent Dye–Stained Gels and Blots) for optimal sensitivity.

The SYPRO Tangerine protein gel stain (S12010, SYPRO(R) Tangerine Protein Gel Stain) is available as 500 µL of a 5000X concentrate in dimethylsulfoxide (DMSO), an amount sufficient to stain about 100 minigels. Significant discounts are available on multiple unit purchases.

Figure 9.29 SYPRO Tangerine protein gel stain and ELF β-D-glucuronide for zymography. Molecular weight standards containing decreasing amounts of Escherichia coli β-glucuronidase were run on an SDS-polyacrylamide gel and stained with SYPRO Tangerine protein gel stain (orange, S12010) followed by incubation with the ELF 97 β-D-glucuronidase substrate (green, E6587) to detect the activity of β-glucuronidase.




Figure 9.30 Linearity of SYPRO Tangerine protein gel stain (S12010). A dilution series of carbonic anhydrase was electrophoresed through a 13% SDS-polyacrylamide gel and stained with SYPRO Tangerine protein gel stain in 7% acetic acid. The fluorescence signal was quantified and plotted versus the protein amount, revealing a sensitivity limit of about 4 ng/band and a linear quantitation range over at least three orders of magnitude.

SYPRO Protein Gel Stain Starter Kit

Our SYPRO Orange, SYPRO Red and SYPRO Tangerine protein gel stains are available in a SYPRO Protein Gel Stain Starter Kit (S12012) for first-time users. Each kit includes:

• 50 µL of SYPRO Orange protein gel stain, sufficient for 5–20 minigels
• 50 µL of SYPRO Red protein gel stain, sufficient for 5–20 minigels
• 50 µL of SYPRO Tangerine protein gel stain, sufficient for 5–20 minigels
• A SYPRO protein gel stain photographic filter
• Detailed protocols (SYPRO(R) Orange and SYPRO(R) Red Protein Gel Stains, SYPRO(R) Tangerine Protein Gel Stain)

Protein Molecular Weight Standards

Molecular Probes offers a protein mixture for use as molecular weight markers in SDS-polyacrylamide gel electrophoresis (Figure 9.31). This broad-range marker mixture (P6649, Protein Molecular Weight Standards) contains a balanced formulation of 11 polypeptides with molecular weights from 6500 to 205,000 daltons. These protein molecular weight standards give rise to sharp, well-separated bands when the gel is stained with any of our protein gel or blot stains. The mixture provides amounts of marker proteins sufficient for loading about 200 gel lanes.

Molecular Probes also has available PeppermintStick phosphoprotein molecular weight standards (P33350), CandyCane glycoprotein molecular weight standards (C21852) and human heart mitochondrial proteins for SDS-polyacrylamide gel electrophoresis (M22430), all of which are described in Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4.

Figure 9.31 Protein molecular weight standards (P6649) separated on a 15% SDS-polyacrylamide gel and stained with SYPRO Orange protein gel stain (S6650, S6651).

Electrophoretic Mobility-Shift (Bandshift) Assay (EMSA) Kit

Molecular Probes has made bandshift assays easy and more convenient with our Electrophoretic Mobility-Shift Assay (EMSA) Kit (E33075). Our EMSA Kit provides a fast and quantitative fluorescence-based method to detect both nucleic acid and protein in the same gel (), doubling the information that can be obtained from bandshift assays. This kit uses two fluorescent dyes for detection — SYBR Green EMSA nucleic acid gel stain for RNA or DNA and SYPRO Ruby EMSA protein gel stain for proteins. Because the nucleic acids and proteins are stained in the gel after electrophoresis, there is no need to prelabel the DNA or RNA with a radioisotope, biotin or a fluorescent dye before the binding reaction, and therefore there is no possibility that the label will interfere with protein binding. Staining takes only about 20 minutes for the nucleic acid stain, and about 4 hours for the subsequent protein stain, yielding results much faster than radioisotope labeling (which may require multiple exposure times) or chemiluminescence-based detection (which requires blotting and multiple incubation steps). This kit also makes it possible to perform ratiometric measurements of nucleic acid and protein in the same band, providing more detailed information on the binding interaction. The signals from the two stains are linear over a broad range, allowing accurate determination of the amount of nucleic acid and protein, even in a single band, with detection limits of ~1 ng for nucleic acids and ~20 ng for protein. Both stains can be detected using a standard 300 nm UV illuminator, a 254 nm epi-illuminator or a laser scanner (). Digital images can easily be overlaid for a two-color representation of nucleic acid and protein in the gel. The EMSA Kit contains sufficient reagents for 10 nondenaturing polyacrylamide minigel assays, including:

• SYBR Green EMSA nucleic acid gel stain
• SYPRO Ruby EMSA protein gel stain
• Trichloroacetic acid, for preparing the working solution of SYPRO Ruby EMSA protein gel stain
• Concentrated EMSA gel-loading solution
• lac repressor, a DNA-binding protein to be used as a control
• lac operator, control DNA
• Concentrated buffer for the lac repressor:operator controls
• A detailed protocol (Electrophoretic Mobility Shift Assay (EMSA) Kit)

Analysis of DNA Structure, DNA Binding and DNA Damage - Section 8.7 describes several other probes and reagents for analyzing DNA structure, DNA binding and DNA damage.

Molecular Probes' proprietary Coomassie Fluor Orange protein gel stain (C33250, C33251) provides fast, simple and sensitive staining of proteins in SDS-polyacrylamide electrophoretic gels. Gels do not need to be washed before staining with the Coomassie Fluor Orange dye or destained after staining — after electrophoresis, the gel is simply stained, rinsed and photographed on a standard UV transilluminator. Our premixed and ready-to-use Coomassie Fluor Orange protein gel stain offers the following advantages over conventional colorimetric stains:

• High sensitivity. Coomassie Fluor Orange protein gel stain detects as little as 8 ng of protein per minigel band (). This sensitivity matches the best colloidal Coomassie stains and handily beats standard Coomassie brilliant blue stains.
• Broad linear range of detection. The fluorescence intensity of Coomassie Fluor Orange dye–stained bands is linear with protein quantity over at least two orders of magnitude, providing accurate quantitation.
• Rapid staining. Staining is complete in less than an hour, and there is no risk of overstaining the gel.
• Compatibility with standard laboratory equipment. Stained proteins can be visualized using a standard 300 nm UV transilluminator or a laser scanner (Figure 9.33). For optimal sensitivity using a UV transilluminator and Polaroid 667 black-and-white print film, the SYPRO photographic filter (S6656, Figure 23.51) is recommended.
• Low protein-to-protein variability. Because Coomassie Fluor Orange dye interacts with the SDS coat around proteins in the gel, it gives more consistent staining between different types of proteins, as compared with Coomassie brilliant blue staining, and it never exhibits negative staining. Coomassie Fluor Orange dye also stains glycoproteins.
• High selectivity for proteins. Coomassie Fluor Orange protein gel stain detects a variety of proteins down to ~6500 daltons without staining nucleic acid or lipopolysaccharide contaminants that are sometimes found in protein preparations derived from cell or tissue extracts.

Coomassie Fluor Orange protein gel stain is not recommended for staining proteins in 2-D, IEF or nondenaturing gels; for these applications we recommend our SYPRO Ruby protein gel stain (see above; S12000, S12001, S21900).

Figure 9.33 Fluorescence excitation and emission spectra of the Coomassie Fluor Orange protein gel stain (C33250) in a solution of 150 µg/mL bovine serum albumin (BSA) with 0.05% SDS.

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 with other gel strengtheners. 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.

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, which is described in Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4. Our Rhinohide Polyacrylamide Gel Strengthener Kit (R33410) includes:

• Rhinohide polyacrylamide gel strengthener
• Premeasured acrylamide/bis-acrylamide mixture (37.5:1 ratio)
• A protocol for casting gels from 5% to 20% (Rhinohide Polyacrylamide Gel Strengthener)

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.

Figure 9.34 Demonstration of the strength and durability of gels made with the Rhinohide polyacrylamide gel strengthener (R33400, R33410).

To characterize specific proteins in complex mixtures, proteins are frequently separated by electrophoresis, then blotted onto nitrocellulose or PVDF (poly(vinylidene difluoride)) membranes (blots) for immunostaining (referred to as Western blotting), glycoprotein staining, sequencing or mass spectrometry. Universal protein stains provide valuable information about the protein samples on blots, making it possible to assess the efficiency of protein transfer to the blot, detect contaminating proteins in the sample and compare the sample with molecular weight standards. For blots of 2-D gels, staining of the entire protein profile makes it easier to localize a protein to a particular spot in the complex protein pattern. The superior properties of our fluorescent and luminescent protein stains, compared with conventional colorimetric stains, makes it possible to quickly and easily obtain this information without running duplicate gels. Combining our luminescent and fluorescent protein staining technology with our fluorescent reagents for selective protein detection, which are described in detail in Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4, creates the capability of multiparameter staining with our Multiplexed Proteomics technology.

SYPRO Ruby Protein Blot Stain: A Versatile Blot Stain

The SYPRO Ruby protein blot stain (Summary of fluorescent and luminescent protein blot stains - Table 9.4) provides fast and highly sensitive detection of proteins blotted onto membranes, making it easy to assess the efficiency of protein transfer to the blot and to determine if lanes are loaded equally (Figure 9.35). Because the stain does not covalently alter the proteins, it can be used to locate proteins on blots before sequencing or mass spectrometry. It can also be used before chromogenic, fluorogenic or chemiluminescent immunostaining procedures to locate molecular weight markers and visualize the total-protein profile in the sample. Furthermore, the stain does not interfere with subsequent identification techniques, eliminating the need to run duplicate gels, vastly simplifying the comparison of total protein and target protein on Western blots, and allowing precise localization of the target protein relative to other proteins on electroblots of 2-D gels. The SYPRO Ruby protein blot stain for general protein detection is also compatible with our Pro-Q Emerald glycoprotein blot stains for glycoproteins (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4, Figure 9.53). The superior properties of the SYPRO Ruby protein blot stain, as compared with conventional protein blot stains, make it possible to routinely stain blots for total protein before continuing with specific protein detection techniques.

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.

The SYPRO Ruby protein blot stain (S11791, SYPRO(R) Ruby Protein Blot Stain) combines the following superior staining characteristics:

• High sensitivity. SYPRO Ruby protein blot stain can detect as little as 0.25–1 ng protein/mm² (~2–8 ng/band) blotted onto PVDF or nitrocellulose membranes after only 15 minutes of staining (Figure 9.35). This sensitivity on blots is far better than that of colorimetric stains, such as Ponceau S, amido black or Coomassie brilliant blue, and rivals the best colloidal gold blot-staining techniques (Figure 9.36).
• Rapid total-protein staining procedure. The SYPRO Ruby protein blot-staining protocol takes less than an hour — including fixation and wash steps — and maximum sensitivity is achieved after only 15 minutes of dye staining, even for some peptides as small as seven amino acids (SYPRO(R) Ruby Protein Blot Stain). In contrast, gold or silver staining procedures may require overnight incubations to achieve maximum sensitivity and usually include extensive wash procedures that must be carefully timed.
• Compatibility with Western blot protocols. Staining the total-protein profile on the blot eliminates guesswork about transfer efficiency and removes the need to run two gels for comparison of total and target protein. The SYPRO Ruby protein blot stain is gentle and, unlike colorimetric or colloidal gold blot stains, does not interfere with subsequent colorimetric or chemiluminescent immunodetection of proteins on Western blots (Figure 9.59, ). The SYPRO Ruby protein blot stain is available as a component of the Pro-Q Glycoprotein Blot Stain Kits and Pro-Q Western Blot Stain Kits (Fluorescence-based Western blot stain kits - Table 9.9). These products are described in detail in Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4.
• Compatibility with standard microsequencing and mass spectrometry protocols. Whereas colloidal gold, Coomassie brilliant blue and amido black staining can interfere with post-staining analysis, SYPRO Ruby protein blot stain binds noncovalently to proteins and is thus fully compatible with Edman sequencing or mass spectrometry.
• Easily visualized signal. The SYPRO Ruby protein blot stain comprises ruthenium complexed with an organic chelator. Because the ruthenium complex has dual-excitation maxima (Figure 9.37), the dye exhibits luminescence upon excitation with either UV or visible light. This property makes it possible to visualize the luminescence with many types of instruments, including UV epi-illumination sources, UV or blue-light transilluminators and a variety of laser-scanning instruments, including those with excitation light at 450 nm, 473 nm, 488 nm or 532 nm. Also, SYPRO Ruby dye–stained proteins can be indirectly excited by the chemiluminescence of the high-energy intermediate produced in the reaction of bis-(2,4,6-trichlorophenyl) oxalate (TCPO) with H₂O₂. The red luminescence of the ruthenium complex has a peak at ~618 nm that is well separated from these excitation peaks, minimizing the amount of background signal seen from the excitation source. The staining signal can be documented using Polaroid 667 black-and-white print film and a SYPRO photographic filter (S6656, Photographic Filters for Fluorescent Dye–Stained Gels and Blots), using a CCD-based imaging system equipped with a 600 nm bandpass or 490 nm longpass filter, or by using the appropriate filter set and software in a laser scanner (Imaging platforms validated as suitable for visualization of SYPRO Ruby protein stains - Table 9.5). The SYPRO Ruby protein blot stain has exceptional photostability, allowing long exposure times for maximum sensitivity.

The Patented SYPRO Ruby protein blot stain (S11791) is supplied as a 1X staining solution, which is sufficient for staining ~1600 cm² of blotting membrane, and is accompanied by a detailed protocol for its use (SYPRO(R) Ruby Protein Blot Stain).

Figure 9.35 Protein detection with SYPRO Ruby protein blot stain (S11791). Samples of protein molecular weight standards containing a twofold dilution series of α-tubulin, starting with 1 µg of tubulin in the left-most lane, were run on an SDS-polyacrylamide gel, blotted onto a PVDF membrane and stained with the SYPRO Ruby protein blot stain.

Figure 9.36 Comparison of commonly used stains for proteins on blots. Twofold serial dilutions of protein molecular weight standards ranging from 2000 to 1 ng/band were run on six identical SDS-polyacrylamide gels and blotted to PVDF membrane. The membranes were then stained with A) Ponceau S stain, B) Coomassie brilliant blue stain, C) colloidal silver stain, D) amido black stain, E) colloidal gold stain or F) SYPRO Ruby protein blot stain (S11791).



Figure 9.37 Luminescence excitation and emission spectra of the SYPRO Ruby protein blot stain (S11791).

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).

SYPRO Rose Plus Protein Blot Stain: A Readily Reversible Protein Blot Stain

Our Patented SYPRO Rose Plus protein blot stain (S12011, SYPRO(R) Rose Plus Protein Blot Stain Kit) has the same high-sensitivity detection capability as the SYPRO Ruby blot stain — about 0.25–1 ng protein/mm² (~2–8 ng/band) — and is fully compatible with subsequent immunostaining, lectin staining, mass spectrometry and Edman sequencing. However, unlike the SYPRO Ruby dye, the SYPRO Rose Plus dye produces protein staining that can be completely reversed by washing the blot. Because the staining can be so easily reversed, SYPRO Rose Plus protein blot stain may be useful for the temporary detection of proteins on other surfaces, like protein arrays, where it would be useful for signal normalization or quality control. It has been used to detect fingerprints on surfaces () and may be useful for detecting cells and proteins on contact lenses, electronic components and other surfaces for quality control purposes.

The SYPRO Rose Plus protein blot stain contains europium as the luminophore. The stain has an excitation maximum at ~350 nm and a narrow emission peak at ~610 nm (Figure 9.39). Stained proteins can be visualized using UV epi-illumination; the excitation characteristics of the SYPRO Rose Plus protein blot stain preclude it from being visualized using visible-wavelength excitation sources. Like the SYPRO Ruby protein blot stain, the SYPRO Rose Plus protein blot stain has exceptional photostability. In addition, the europium luminescence has a very long emission lifetime (20–50 µsec), which may allow time-resolved luminescence measurements that would greatly minimize background fluorescence.

The SYPRO Rose Plus protein blot stain (S12011) is provided as a kit containing:

• SYPRO Rose Plus blot stain solution
• SYPRO Rose Plus blot wash solution
• SYPRO Rose Plus blot destain solution
• A detailed protocol (SYPRO(R) Rose Plus Protein Blot Stain Kit)

The quantities of reagents are sufficient to stain ~1600 cm² of blotting membrane.

Figure 9.39 Luminescence excitation and emission spectra of the SYPRO Rose Plus protein blot stain, a component of the SYPRO Rose Plus Protein Blot Stain Kit (S12011).

Detection of Proteins on Protein Microarrays

We have found that our protein blot stains perform particularly well when staining proteins on PVDF microarrays for quality control and normalization purposes. SYPRO Ruby protein blot stain (S11791) shows good sensitivity on protein microarrays () and should be very useful for staining proteins before exposing the microarray to the sample. The stain washes off PVDF membranes very easily under conditions used with typical Western blot blocking buffers. SYPRO Rose Plus protein blot stain (S12011) may also be useful for the temporary detection and normalization of proteins on microarrays because the staining is so easily reversed. BODIPY FL-X succinimidyl ester (D6102) shows even greater sensitivity in this application, as described below, and should be useful for quality control or as an internal normalization standard.

Reactive Fluorescent Dyes for Permanent Protein Blot and Microarray Staining

Our Patented BODIPY reactive dyes in BODIPY Dye Series - Section 1.4 label amine groups (predominantly lysine residues) on proteins, and we have found the BODIPY FL-X succinimidyl ester and BODIPY TR-X succinimidyl ester (D6102, D6116) to be particularly effective general stains for proteins on PVDF membranes (Summary of fluorescent and luminescent protein blot stains - Table 9.4). This unique method of staining for total protein on blots with the reactive BODIPY dyes has an approximately 30-fold linear dynamic range (Figure 9.64), although the absolute intensity between proteins may vary somewhat with the nature of the protein. Reactive BODIPY dye–based staining is rapid, simple and highly sensitive, permitting detection of as little as 4 ng of a protein per band in about an hour. Because the reactive dyes form a covalent bond with the protein, the staining is permanent and lasts through any subsequent conventional blot manipulations. The covalent modification appears to minimally interfere with subsequent immunostaining, as we have successfully performed simultaneous two-color labeling with reactive BODIPY dyes and either fluorogenic immunostains or fluorogenic glycoprotein stains and found both stains to be visible at the same time on the same blot (, Figure 9.63). Simultaneous dual labeling of a sample enormously simplifies localization of a specific protein with respect to other proteins in the sample, particularly on electroblots of 2-D gels, pairs of which are difficult to align.

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 9.63 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).

The optimal BODIPY dyes and procedures for use with our fluorogenic Western blot detection reagents are included in our DyeChrome Western Blot Stain Kits (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4, Fluorescence-based Western blot stain kits - Table 9.9). The green-fluorescent BODIPY FL-X succinimidyl ester is used in combination with DDAO phosphate (Figure 9.63, Figure 9.66), which produces a red-fluorescent product in the presence of alkaline phosphatase; the red-fluorescent BODIPY TR-X succinimidyl ester is used in combination with ELF 39 phosphate (, Figure 9.67), which produces a green-fluorescent product in the presence of alkaline phosphatase. The fluorescent staining can be visualized using UV illumination or a laser scanner. The stains can be documented simultaneously using color photography or — using the appropriate filters, such as those in the DyeChrome Red/Green Photographic Filter Set (D24771; Accessories for Electrophoresis - Section 23.4; Figure, Figure) — can be documented separately. Note that because reaction of the dye covalently modifies the protein at random locations, staining by the amine-reactive BODIPY dye may complicate or preclude subsequent analysis by mass spectrometry or microsequencing.

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).

Our DyeChrome Double Western Blot Stain Kit (D21887, Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4) uses another fluorescent reactive dye, MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone), to stain the total-protein profile on PVDF membranes. This blue-fluorescent dye is visible using UV epi-illumination and can be used with two (or possibly more) different fluorogenic Western blot stains. In the DyeChrome Double Western Blot Stain Kit, MDPF is used together with DDAO phosphate, which produces the red-fluorescent DDAO dye (Figure 10.7, ) in the presence of alkaline phosphatase, and our proprietary Amplex Gold reagent, which produces a yellow-fluorescent compound in the presence of horseradish peroxidase, for detection of two different specific proteins and the total-protein profile, all on the same blot (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4, Figure 9.70).

Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit

The Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit (P33706) provides a method for selective staining of phosphoproteins or phosphopeptides on microarrays, without the use of antibodies or radioactivity. This kit permits direct detection of phosphate groups attached to tyrosine, serine or threonine residues in a microarray environment and has been optimized for microarrays with acrylamide gel surfaces. Each Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit provides:

• Pro-Q Diamond phosphoprotein/phosphopeptide microarray stain
• Pro-Q Diamond microarray destain solution
• Microarray staining gasket with seal tabs, 10 chambers
• Slide holder tube, 20 tubes
• Detailed protocols (Pro-Q(R) Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit)

The Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit is ideal for identifying kinase targets in signal transduction pathways and for phosphoproteomics studies.

Capillary electrophoresis (CE) is an exceptionally powerful tool for the resolution of biomolecules. Fluorescent detection of proteins that are separated by capillary electrophoresis can occur either during the run — the more common procedure — or subsequent to the separation on isolated fractions. When detected during the electrophoretic separation, the protein is either derivatized with a fluorescent reagent prior to the separation or labeled with a fluorescent dye that is incorporated into the running medium. In general, the same reagents may be useful for fluorometric detection of peptides and proteins that are separated by high-performance liquid chromatography (HPLC). In addition the total-protein staining techniques described below, many selective staining techniques, such as the use of BODIPY FL GTP-γ-S to detect GTP-binding proteins, can be applied to proteins separated by capillary electrophoresis.

Use of SYPRO Dyes for Capillary Electrophoresis

SDS–capillary gel electrophoresis (SDS-CGE) separates proteins based on principles similar to those of standard slab-gel electrophoresis, but with the advantages of faster run times, higher resolution and greater sensitivity. The use of online detection by laser-induced fluorescence (LIF) increases the sensitivity several orders of magnitude over UV detection, eliminates the time spent staining and photographing the gel and allows for the possibility of automated sample processing. SDS-CGE analyzed by LIF is already being widely used for the separation and identification of DNA fragments and has increased the efficiency of genomics, DNA typing and forensics laboratories. SDS-CGE promises to be just as useful for proteomics laboratories and other laboratories that require characterization of a large number of protein samples.

For SDS-CGE of protein samples, the amine groups of lysine residues and the N-terminus of the proteins are typically derivatized with a fluorescent or fluorogenic dye such as the ATTO-TAG CBQCA (A2333, A6222) or ATTO-TAG FQ (A2334, A10192) reagents before separation in the capillary. The derivatized proteins are then coated with SDS and travel through the capillary gel towards the positive electrode based on their size, with smaller proteins traveling faster. The derivatized proteins are detected by fluorescence emitted as they pass a laser that excites the fluorophores. One disadvantage of this technique is that the proteins generally contain multiple amine groups, each of which can react with the derivatization reagent. Typically, only a few of the amine groups on each protein molecule react, and the result is an enormous number of different derivatives, creating broad peaks that may be difficult to correlate with the original protein's structure or abundance. In addition, variations between runs make it difficult to reproducibly estimate molecular weights. In contrast, use of SYPRO Red protein gel stain (S6653, S6654) to prestain SDS-coated proteins allows more accurate determination of molecular weights because the proteins are relatively uniformly coated with SDS and the dye. This method leads to molecular weight determinations similar in accuracy to those achieved with polyacrylamide slab gels, with a limit of detection estimated to exceed the detection limit of silver staining in slab gels.

Use of SYTO, SYBR and NanoOrange Dyes for Capillary Electrophoresis

Our SYTO and SYBR dyes — which are extremely useful stains for nucleic acids (Nucleic Acid Detection and Genomics Technology - Chapter 8) — are essentially nonfluorescent in aqueous medium unless they are bound to nucleic acids. However, we have found that these same dyes may become highly fluorescent once bound to lipid-complexed proteins. Although we are not aware of any published description of the use of these dyes for detecting proteins in capillary electrophoresis, we anticipate that they can be used in the same manner as the SYPRO dyes for these applications. The broad spectral range of available SYTO (Cell-permeant cyanine nucleic acid stains - Table 8.3) and SYBR dyes (Specialty nucleic acid reagents for molecular biology - Table 8.1) should permit their use with a wide variety of excitation sources. Several of the SYTO and SYBR dyes also stain proteins in SDS-polyacrylamide gels, but usually with lower sensitivity than do the SYPRO dyes. The NanoOrange reagent (N6666, Quantitation and Selective Purification of Proteins in Solution - Section 9.2) is reported to be an optimal reagent for detecting proteins that have been separated by microchip capillary electrophoresis.

Derivatization Reagents for Proteins

Several of the same reagents that were described in Quantitation and Selective Purification of Proteins in Solution - Section 9.2 for protein quantitation in solution are also useful for peptide and protein derivatization, either prior to or following separation by capillary electrophoresis. However, chemical derivatization prior to separation is likely to change the electronic charge and always changes the mass of the protein. Furthermore, incomplete derivatization of amines or thiols on the protein can lead to a pure protein resolving into multiple species in the electrophoretogram.

In an improved procedure for fluorescent analysis of peptides by capillary electrophoresis, Zhou and colleagues modified all α- and ε-amino groups of the peptide with phenyl isothiocyanate. Following one cycle of Edman degradation, the single free α-amino group was modified with fluorescent reagents to give a homogeneous, dye-labeled peptide.

The preferred reagents for derivatizing amine residues in proteins either prior to or following electrophoretic separation are those that are essentially nonfluorescent until reacted with the protein. Derivatization reagents that react with thiols or other functional groups have also been used. These preferred reagents include:

• ATTO-TAG CBQCA, which is available in our ATTO-TAG CBQCA Amine-Derivatization Kit (A2333) and as a stand-alone reagent (A6222). ATTO-TAG CBQCA reacts with primary amines to form highly fluorescent isoindoles and has been extensively used for the derivatization of amino acids, peptides and carbohydrates prior to capillary electrophoretic separation. ATTO-TAG CBQCA has been used to derivatize a fusion protein expressed in the bacterium Escherichia coli before purification by capillary zone electrophoresis. After purification, the fluorescent isoindole can be removed by acid treatment to allow sequencing of the purified protein.
• ATTO-TAG FQ (3-(2-furoyl)quinoline-2-carboxaldehyde), which is available in our ATTO-TAG FQ Amine-Derivatization Kit (A2334) and as a stand-alone reagent (A10192). ATTO-TAG FQ has been used as a protein detection reagent in capillary electrophoresis. It has been reported that ATTO-TAG FQ can detect as little as 200 attomoles of a protein by capillary electrophoresis. Excitation of amine derivatives of ATTO-TAG FQ by the 488 nm spectral line of the argon-ion laser is more efficient than that of ATTO-TAG CBQCA derivatives. A report describes the solid-phase derivatization of dilute peptide solutions (10^-8 M) that have been immobilized on Immobilon CD membranes. This technique permits the quantitative derivatization and analysis by capillary electrophoresis of only a few picomoles of the analyte.
• Fluorescamine (F2332; FluoroPure Grade - Note 19.2, F20261), a nonfluorescent reagent that rapidly reacts with amines to give a fluorescent product. Fluorescamine has been used for solution quantitation of proteins and peptides (Quantitation and Selective Purification of Proteins in Solution - Section 9.2). It is also useful as a peptide and protein detection reagent for capillary electrophoresis. Use of fluorescamine to derivatize a standard protein of known molecular weight together with use of the ATTO-TAG FQ reagent to derivatize the sample protein allows the sample to be run simultaneously with the standard, improving the accuracy of molecular weight determination. Chiral separation of fluorescamine-labeled amino acids has been optimized using capillary electrophoresis in the presence of hydroxypropyl-β-cyclodextran, a method designed for use in extraterrestrial exploration on Mars.
• Dialdehydes OPA and NDA (P2331MP, N1138), which react with amines in the presence of a nucleophile (Figure 1.116, Figure 1.117) to give fluorescent products. These inexpensive reagents have been used for capillary electrophoresis of peptides and proteins.
• Other amine-reactive reagents. Fluorophores and Their Amine-Reactive Derivatives - Chapter 1 describes a variety of other amine-reactive reagents, including our numerous Succinimidyl Esters, Isothiocyanates and Sulfonyl Chlorides, that have been used or may be useful for peptide and protein detection in capillary electrophoresis, including dansyl chloride (D21), NBD chloride (FluoroPure Grade - Note 19.2, C20260), NBD fluoride (F486), FITC (F143) and other common reagents described in Reagents for Analysis of Low Molecular Weight Amines - Section 1.8. After reaction with Alexa Fluor succinimidyl esters (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum - Section 1.3), proteins in bacteriophage T7 capsid have been separated and detected in gels by their intrinsic fluorescence; analyzing protein accessibility with the water-soluble Alexa Fluor dyes can also be applied to the localization of proteins in cells and tissues.
• Reactive reagents for other groups on proteins. Thiol-reactive probes such as Maleimides and Iodoacetamides can be used for the selective detection of natural or engineered proteins that contain a free thiol group (cysteine). Most of the fluorescent derivatization reagents for thiols (Thiol-Reactive Probes - Chapter 2) could potentially be used for detection of thiolated proteins in capillary electrophoresis. Thiol-reactive reagents that are essentially nonfluorescent until conjugated to thiols, such as the coumarin maleimides CPM and DACM (D346, D10251; Thiol-Reactive Probes Excited with Ultraviolet Light - Section 2.3), monobromobimane (M1378; FluoroPure Grade - Note 19.2, M20381; Thiol-Reactive Probes Excited with Ultraviolet Light - Section 2.3) and N-(1-pyrene)maleimide (P28, Thiol-Reactive Probes Excited with Ultraviolet Light - Section 2.3), should work well in this application. Although intrinsically fluorescent, BODIPY iodoacetamides and maleimides (Thiol-Reactive Probes Excited with Visible Light - Section 2.2) have been used to detect thiol-containing proteins in SDS gels and by reverse-phase HPLC. Proteins that have been labeled with 5-iodoacetamidofluorescein (I30451, Thiol-Reactive Probes Excited with Visible Light - Section 2.2) have been analyzed by capillary electrophoresis. A particularly unique derivatization scheme using 6-iodoacetamidofluorescein (I30452, Thiol-Reactive Probes Excited with Visible Light - Section 2.2) has been applied to selective detection of peptides and proteins containing phosphoserine residues by capillary electrophoresis (Figure 9.41). Although the original procedure used 6-iodoacetamidofluorescein, almost any of the thiol-reactive reagents in Thiol-Reactive Probes - Chapter 2 may be useful for this method. To identify glycoproteins in capillary electrophoresis, reagents derived from Hydrazine Derivatives and Hydroxylamine Derivatives such as those described in Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2 may be useful for indirectly labeling the hydroxyl groups subsequent to their oxidation to aldehydes. Dansyl hydrazine (D100, Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2) labeling has been used to detect periodate-oxidized proteins separated by HPLC.

Figure 9.41 Selective detection of phosphoserine residues in proteins via derivatization by 1,2-ethanedithiol, followed by 6-iodoacetamidofluorescein (I30452).