Quantitation and Selective Purification of Proteins in Solution - Section 9.2

The Quant-iT family of assay kits provides state-of-the-art reagents for sensitive and selective quantitation of protein (A comparison of reagents for detecting and quantitating proteins in solution - Table 9.1), DNA or RNA (Selection Guide for the Quant-iT Assay Kits - Table 8.12) samples using a standard fluorescence microplate reader. These kits have been specially formulated with ready-to-use buffers, prediluted standards and easy-to-follow instructions, making quantitation both accurate and extremely easy (Figure 8.52). Each Quant-iT assay is:

• Ready to use. Only the dye is diluted in the supplied buffer; no dilution of standards or buffer required.
• Easy to perform. Just add the sample to the diluted dye and read the fluorescence.
• Highly sensitive. The Quant-iT protein assay is orders of magnitude more sensitive than UV absorbance measurements.
• Highly selective. Separate kits are available for quantitating DNA, RNA (Nucleic Acid Detection and Quantitation in Solution - Section 8.3) or protein (see below), with minimal interference from common contaminants.
• Precise. CVs are generally less than 5% for typical users.

Because the fluorescent dye in each Quant-iT Kit matches common fluorescence excitation and emission filter sets in microplate readers, these assay kits are ideal for high-throughput environments, as well as for small numbers of samples.

The Quant-iT Protein Assay Kit (Q33210) simplifies protein quantitation without sacrificing sensitivity. This protein assay exhibits a detection range between 0.25 and 5 µg protein (Figure 9.3), and the response curve is sigmoidal (pseudolinear from 0.5 to 4 µg) with little protein-to-protein difference in signal intensity. Common contaminants, including salts, solvents, 2-mercaptoethanol, amino acids and DNA, are well tolerated in this assay; however, it is not compatible with detergents. Each Quant-iT Protein Assay Kit contains:

• Quant-iT protein reagent
• Quant-iT protein buffer
• A set of eight prediluted bovine serum albumin (BSA) standards between 0 and 500 ng/µL
• Easy-to-follow instructions (Quant-iT Protein Assay Kit)

Sufficient reagents are provided to perform 1000 assays, based on a 200 µL assay volume in a 96-well microplate format; this assay can also be adapted for use in cuvettes or 384-well microplates. The fluorescence signal exhibits excitation/emission maxima of 470/570 nm and is stable for three hours at room temperature. The Quant-iT protein reagent is a new formulation of Molecular Probes' NanoOrange reagent, which is described below.

Figure 9.3 Low protein-to-protein variation in the Quant-iT protein assay. Solutions of the following proteins were prepared, diluted and assayed with the Quant-iT Protein Assay Kit (Q33210): bovine serum albumin (BSA), chicken-egg ovalbumin, chicken-egg lysozyme, bovine-milk β-casein, equine myoglobin, bovine-milk α-casein, porcine pepsin, mouse immunoglobulin (IgG) and calf-thymus histone. Fluorescence was measured at 485/590 nm and plotted versus the mass of protein sample. At 3 µg, the fluorescence variation was 12.4%, or 8.7% excluding the highly basic histone protein. Background fluorescence has not been subtracted.

Our Patented NanoOrange Protein Quantitation Kit (N6666) provides an ultrasensitive assay for measuring the concentration of proteins in solution. The NanoOrange Protein Quantitation Kit has several important features:

• Ease of use. The NanoOrange assay protocol is much easier to perform than the Lowry method (Figure 9.4). Protein samples are simply added to the diluted NanoOrange reagent in a lipid-containing medium, and the mixtures are heated at 95°C for 10 minutes. After cooling the mixtures to room temperature, their fluorescence emissions are measured directly. The interaction of the lipid-coated proteins with the NanoOrange reagent produces a large fluorescence enhancement that can be used to generate a standard curve for protein determination; fluorescence of the reagent in aqueous solutions in the absence of proteins is negligible.
• Sensitivity and effective range. The NanoOrange assay can detect proteins at a final concentration as low as 10 ng/mL when a standard spectrofluorometer or minifluorometer is used. A single protocol is suitable for quantitating protein concentrations between 10 ng/mL and 10 µg/mL — an effective range of three orders of magnitude (Figure 9.5).
• Stability. The NanoOrange reagent and its protein complex have high chemical stability. In contrast to the Bradford and BCA assays, readings can be taken for up to six hours after sample preparation with no loss in signal, provided that samples are protected from light.
• Little protein-to-protein variability (Figure 9.6). The NanoOrange assay is not only more sensitive, but shows less protein-to-protein variability than Bradford assays.
• Insensitivity to sample contaminants. Unlike the Lowry and BCA assays, the NanoOrange assay is compatible with the presence of reducing agents. Furthermore, the high sensitivity of the assay and stability of the protein–dye complex make it possible to dilute out most potential contaminants, including detergents and salts (Tolerance levels for contaminants in the NanoOrange protein quantitation assay - Table 9.2). Nucleic acids do not interfere with protein quantitation using the NanoOrange reagent. Although unusually high concentrations of lipids in the sample can interfere with the NanoOrange assay, this interference can be eliminated by acetone precipitation of the protein, followed by delipidation with diethyl ether.

Our NanoOrange protein quantitation reagent, with an excitation/emission maxima of 470/570 nm when bound to proteins, is suitable for use with a variety of instrumentation. Fluorescence is typically measured using instrument settings or filters that provide excitation/emission at ~485/590 nm, which are commonly available for both spectrofluorometers and microplate readers. A spectrofluorometer — either a standard fluorometer or a minifluorometer — offers the greatest effective range and lowest detection limits for this assay. With fluorescence microplate readers, the NanoOrange assay is useful over a somewhat narrower range — from 100 ng/mL to 10 µg/mL in final protein concentration.

The NanoOrange Protein Quantitation Kit (N6666) supplies:

• Concentrated NanoOrange reagent in dimethylsulfoxide (DMSO)
• Concentrated NanoOrange diluent
• Bovine serum albumin (BSA) as a protein reference standard
• A detailed protocol for protein quantitation (NanoOrange(R) Protein Quantitation Kit)

The amount of dye supplied in this kit is sufficient for ~200 assays using a 2 mL assay volume and a standard fluorometer or minifluorometer, or ~2000 assays using a 200 µL assay volume and a fluorescence microplate reader.

The NanoOrange reagent is ideal for quantitating protein samples before gel electrophoresis and Western blot analysis. It has also been used to measure bound versus free protein levels in protein binding assays, and was even able to detect protein trapped in filters during a separation step. The NanoOrange reagent is also an optimal reagent for detecting proteins that have been separated by microchip capillary electrophoresis. A high-throughput assay that may be suitable for clinical samples has been developed for quantitating human serum albumin using a fluorescence microplate reader and using capillary electrophoresis laser-induced fluorescence (CE-LIF). Additionally, the NanoOrange reagent has been shown to be useful in cell-based assays, including an assay designed to measure total protein content of cell cultures and a rapid method for demonstrating flagellar movement of bacteria.

Figure 9.5 Quantitative analysis of bovine serum albumin (BSA) using the NanoOrange Protein Quantitation Kit (N6666). Fluorescence measurements were carried out on an SLM SPF-500C fluorometer using excitation/emission wavelengths of 485/590 nm. The inset shows an enlargement of the results obtained (0–500 ng protein per mL) and illustrates the detection limit of ~10 ng/mL.

Figure 9.6 Quantitative analysis of six different proteins using the NanoOrange Protein Quantitation Kit (N6666): A) bovine serum albumin (BSA, ), trypsin () and carbonic anhydrase (); B) IgG (), streptavidin () and RNase A (). The y-axis fluorescence intensity scale is the same in both panels, illustrating the minimal protein-to-protein staining variation of the NanoOrange assay. Data were collected using a microplate reader with excitation/emission wavelengths set at 485 ± 20 nm/590 ± 35 nm.

The ATTO-TAG CBQCA reagent was originally developed as a chromatographic derivatization reagent for amines (Reagents for Analysis of Low Molecular Weight Amines - Section 1.8), but this reagent is also useful for quantitating proteins (Figure 9.7) by virtue of its rapid and quantitative reaction with their accessible amines. Molecular Probes has developed the CBQCA Protein Quantitation Kit (C6667, Figure 9.8), which employs the ATTO-TAG CBQCA reagent for rapid and sensitive protein quantitation in solution (A comparison of reagents for detecting and quantitating proteins in solution - Table 9.1). The CBQCA protein quantitation assay functions well in the presence of lipids and detergents, substances that interfere with many other protein determination methods. For example, the CBQCA-based assay can be used directly to determine the protein content of lipoprotein samples or lipid–protein mixtures (Figure 9.9). The CBQCA assay has been shown to give faster and more sensitive detection of both free amino acids in human plasma and both low and high molecular weight primary amines in clinical samples from hemodialysis. ATTO-TAG CBQCA is more water soluble than either fluorescamine or o-phthaldialdehyde and much more stable in aqueous solution than fluorescamine. Moreover, ATTO-TAG CBQCA provides greater sensitivity for protein quantitation in solution than either fluorescamine or o-phthaldialdehyde (Figure 9.10). As little as 10 ng of BSA can be detected in a 100–200 µL assay volume using a fluorescence microplate reader, and the effective range extends up to 150 µg (Figure 9.7). Alternatively, the reaction mixtures can be diluted to 1–2 mL for fluorescence measurement in a standard fluorometer or minifluorometer.

Each CBQCA Protein Quantitation Kit (C6667) contains:

• ATTO-TAG CBQCA detection reagent
• Potassium cyanide
• Dimethylsulfoxide (DMSO)
• Bovine serum albumin (BSA) protein reference standard
• A detailed protocol for protein quantitation (CBQCA Protein Quantitation Kit)

The CBQCA Protein Quantitation Kit provides sufficient reagents for 300–800 assays using a standard fluorometer, minifluorometer or fluorescence microplate reader.

Figure 9.7 Detection of bovine serum albumin (BSA) using the CBQCA Protein Quantitation Kit (C6667). The primary plot shows detection of BSA from 50 ng to 1000 ng. Inset A shows that the detection range can extend up to 150 µg. Inset B shows that the lower detection limit can extend down to 10 ng. Each point is the average of four determinations.

Figure 9.8 Protein quantitation with the CBQCA Protein Quantitation Kit. The CBQCA assay (C6667) is simple to perform: after the dye and an activator are added, the sample is incubated for an hour and the fluorescence is read in either a microplate reader or a fluorometer.

Figure 9.9 Quantitation of proteins in a lipid environment using the CBQCA Protein Quantitation Kit (C6667). The protein concentrations of an LDL preparation and a bovine brain homogenate were first determined by the modified Lowry method using BSA as a standard. Assays were then performed using the CBQCA Protein Quantitation Kit on samples containing from 100 ng to 1000 ng protein in 0.1 M sodium borate buffer, pH 9.3, containing 0.1% Triton X-100. Similar results were obtained without the addition of detergent (data not shown). Fluorescence was measured using a fluorescence microplate reader with excitation at 485 ± 10 nm and emission detection at 530 ± 12.5 nm. Each point is the average of three determinations.

Figure 9.10 Comparison of the fluorometric quantitation of bovine serum albumin (BSA) using ATTO-TAG CBQCA (which is supplied in the CBQCA Protein Quantitation Kit, C6667), OPA (P2331MP) or fluorescamine (F2332, F20261). BSA samples were derivatized using large molar excesses of the fluorogenic reagents and were analyzed using a fluorescence microplate reader. Excitation/emission wavelengths were 360/460 nm for OPA and fluorescamine and 485/530 nm for ATTO-TAG CBQCA. The inset shows an enlargement of the results obtained using CBQCA to assay protein concentrations between 0 and 500 ng/mL.

The EZQ Protein Quantitation Kit (R33200) provides a fast and easy high-throughput assay for proteins in solution. Because detergents, reducing agents, urea and tracking dyes do not interfere, this fluorescence-based protein quantitation assay is ideal for determining the protein concentration of samples prior to polyacrylamide gel electrophoresis. This convenient kit can also provide a quick assessment of protein content during protein purification schemes and fractionation procedures.

The EZQ assay requires only 1 µL of a sample per spot, and up to 96 samples, including standards, can be assayed in one session. The protein samples are simply spotted onto one of the provided assay papers, fixed with methanol and then stained with our proprietary EZQ protein quantitation reagent. This assay paper is then clamped into the specially designed 96-well microplate for quick analysis in a top- or bottom-reading fluorescence microplate reader (Figure 9.11). For added versatility, the solid-phase assay format and provided 96-well microplate are also compatible with laser scanners equipped with 450, 473 or 488 nm lasers and with UV illuminators in combination with photographic or CCD cameras for image documentation and analysis. Once the samples are spotted, the assay protocol can be completed in about 1 hour. The protein concentration is determined from a standard curve, and the effective range for the assay is generally 0.05–5 mg/mL or 0.05–5 µg per spot (Figure 9.12). Protein-to-protein sensitivity differences in the assay are minimal — the observed coefficient of variation is typically ~16% (Figure 9.13). The EZQ Protein Quantitation Kit is extremely useful for estimating the concentration of chromatographically separated protein fractions.

Each EZQ Protein Quantitation Kit contains:

• EZQ protein quantitation reagent
• A bottomless 96-well microplate with a stainless steel backing plate
• Assay paper
• Ovalbumin, for preparing protein standards
• A detailed protocol for protein quantitation using a variety of fluorescence-detection instruments (EZQ(R) Protein Quantitation Kit)

Sufficient reagent and assay paper are provided for ~2000 protein quantitation assays.

Figure 9.11 Schematic diagram of method used in the EZQ Protein Quantitation Kit (R33200).

Figure 9.12 EZQ protein quantitation assay of ovalbumin. A dilution series of ovalbumin was prepared, assayed with the EZQ Protein Quantitation Kit (R33200) and then quantitated using both a 473 nm laser–based scanning instrument (upper panel) and a fluorescence microplate reader (lower panel). The assays were performed over a broad range; the insets show the low range in greater detail. The assays were performed in triplicate, and the mean values, in arbitrary fluorescence units, were plotted after subtracting background values of 86 (upper panel) or 18 (lower panel).

Figure 9.13 Protein-to-protein variation in the EZQ protein quantitation assay. Triplicate 1 µg samples of various proteins were assayed using the EZQ Protein Quantitation Kit (R33200) and a fluorescence microplate reader. The mean fluorescence values, after correcting for background fluorescence, are expressed relative to that of ovalbumin. The coefficient of variation is ~16%. The protein samples are: A, ovalbumin; B, bovine serum albumin (BSA); C, myoglobin; D, soybean trypsin inhibitor; E, β-casein; F, carbonic anhydrase; G, transferrin; H, mouse IgG; I, lysozyme; and J, histones.

Other than our premier protein quantitation products described above, most other fluorogenic reagents for general protein quantitation in solution detect accessible primary amines. The sensitivity of assays based on these reagents therefore depends on the number of amines available — a function of both the protein's three-dimensional structure and its amino acid composition. For example, horseradish peroxidase (MW ~40,000 daltons), which has only six lysine residues, will be detected less efficiently than egg white avidin (MW ~66,000 daltons), which has 36 lysine residues, and bovine serum albumin (MW ~66,000 daltons), which has 59 lysine residues. However, the assays are generally rapid and easy to conduct, particularly in minifluorometer and fluorescence microplate reader formats.

Certain dyes that detect primary aliphatic amines, including ATTO-TAG CBQCA (A6222), fluorescamine (F2332; FluoroPure Grade - Note 19.2, F20261) and o-phthaldialdehyde (OPA, P2331MP), have been the predominant reagents for fluorometric determination of proteins in solution (A comparison of reagents for detecting and quantitating proteins in solution - Table 9.1). These same reagents, and others such as naphthalene-2,3-dicarboxaldehyde (NDA, N1138; Reagents for Analysis of Low Molecular Weight Amines - Section 1.8), have frequently been used for amino acid analysis of hydrolyzed proteins.

Fluorescamine

Fluorescamine (F2332; FluoroPure Grade - Note 19.2, F20261) is intrinsically nonfluorescent but reacts in milliseconds with primary aliphatic amines, including peptides and proteins, to yield a fluorescent derivative (Figure 1.115). This amine-reactive reagent has been shown to be useful for determining protein concentrations of aqueous solutions and for measuring the number of accessible lysine residues in proteins. Protein quantitation with fluorescamine is particularly well suited to a minifluorometer or fluorescence microplate reader. Fluorescamine can also be used to detect proteins in gels and to analyze low molecular weight amines by TLC, HPLC and capillary electrophoresis.

o-Phthaldialdehyde

The combination of o-phthaldialdehyde (OPA, P2331MP) and 2-mercaptoethanol provides a rapid and simple method of determining protein concentrations in the range of 0.2 µg/mL to 25 µg/mL (Figure 1.116). As compared with fluorescamine, OPA is both more soluble and stable in aqueous buffers and its sensitivity for detection of peptides is reported to be 5–10 times better. The OPA assay for lysine content is reasonably reliable over a broad range of proteins. OPA (and likely the ATTO-TAG CBQCA reagent) can also be used to detect increases in the concentration of free amines that result from protease-catalyzed protein hydrolysis.

SYPRO Red and SYPRO Orange Protein Gel Stains

An assay has been reported that uses the SYPRO Red protein gel stain (S6653, S6654; Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3) for quantitating total protein content of bacterial cells by flow cytometry. This assay provides an accurate measure of planktonic bacterial biomass in marine samples. Fluorescence of the SYPRO Orange protein gel stain (S6650, S6651; Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3) has been used to follow isothermal protein denaturation and to selectivly stain proteins in biofilms prior to two-photon laser-scanning microscopy.

EZQ Phosphoprotein Quantitation Kit

The EZQ Phosphoprotein Quantitation Kit (E33201) provides a fast and simple assay for phosphoproteins in solution. No radioactivity or antibodies are required, and sample analysis can typically be completed within 60 minutes. The EZQ phosphoprotein quantitation assay shows high selectivity for phosphoproteins over nonphosphorylated proteins (Figure 9.14) and is compatible with samples containing detergents, reducing agents and urea buffers with up to 1% carrier ampholytes. Furthermore, this assay requires only 1 µL of sample, and up to 96 samples, including standards, can be assayed simultaneously. This kit is ideal for analyzing protein kinase or phosphatase activities, as well as for monitoring relative phosphoprotein concentrations during chromatography or after IEF fractionation of protein samples.

In this assay, the phosphoprotein samples are spotted onto specially prepared assay paper, fixed onto the paper with methanol and then stained with our proprietary EZQ phosphoprotein quantitation reagent. Relative phosphate content is determined from a standard curve of ovalbumin or any standard phosphoprotein of interest. As little as 20 ng of ovalbumin that contains 2 phosphate residues per ovalbumin can be selectively detected, and the ovalbumin standard curve has an overall dynamic range of 250-fold, from 0.4 to 120 picomoles. The Z-factor for this assay is in the "excellent" range at greater than 0.8, with N = 8. For normalizing the phosphoprotein signal to the total protein levels, total protein quantitation can be easily performed after phosphoprotein analysis on the same paper using the EZQ Protein Quantitation Kit (R33200) described above. Each EZQ Phosphoprotein Quantitation Kit contains:

• EZQ phosphoprotein quantitation reagent
• EZQ phosphoprotein destain reagent
• EZQ 96-well microplate cassette
• Assay paper
• Ovalbumin standard
• A detailed protocol for phosphoprotein quantitation (EZQ(R) Phosphoprotein Quantitation Kit)

Sufficient materials are provided for 2000 microplate-well assays. The EZQ Phosphoprotein Quantitation Kit is designed for high-throughput analysis. The solid-phase format and special 96-well microplate can be used with readily available fluorescence-based detection instruments, including either top- or bottom-reading microplate readers and laser scanners equipped with 532–560 nm lasers, as well as UV illuminators in combination with photographic or CCD cameras for image documentation and analysis (with lower sensitivity).

Figure 9.14 Highly selective staining of phosphoproteins using the EZQ Phosphoprotein Quantitation Kit (E33201). For each of the proteins assayed, a solution of 1 µg/µL was prepared. One microliter of each solution was spotted onto the assay paper, and the proteins were stained using the kit protocol (Product Information Sheet). Only the phosphorylated proteins (β-casein, ovalbumin and pepsin) show a significant level of staining with respect to the buffer blank. (For the proteins tested, BSA, STI and CA stand for bovine serum albumin, soybean trypsin inhibitor and carbonic anhydrase, respectively.)

EZQ Phosphopeptide Quantitation Kit

The EZQ Phosphopeptide Quantitation Kit (E33202) permits accurate quantitation of phosphopeptides in solution, in the presence of standard buffer components. As with the EZQ Phosphoprotein Quantitation Kit described above, no radioactivity or antibodies are required, and sample analysis can typically be completed within 60 minutes. This phosphopeptide assay requires only 1 µL of sample, and up to 96 samples, including standards, can be assayed simultaneously. This kit is ideal for analyzing phosphatase and kinase activities, as well as for monitoring relative phosphopeptide concentrations before and after analysis by liquid chromatography, mass spectrometry or other separation technique.

In the EZQ phosphopeptide quantitation assay, the phosphopeptide samples are spotted onto specially prepared assay paper, fixed onto the paper with methanol and then stained with our proprietary EZQ phosphopeptide quantitation reagent. Relative phosphate content is determined from a standard curve of control phosphopeptide pT1721 or any standard phosphopeptide of interest. Subpicomole amounts of most monophosphorylated peptides can be detected; for peptides that we have tested, the overall dynamic range of detection is over 500-fold, from 0.2–0.8 picomole at the lower end to about 400 picomoles, depending on the peptide. The Z-factor for this assay is in the "excellent" range at greater than 0.8, with N = 8. Each EZQ Phosphopeptide Quantitation Kit contains:

• EZQ phosphopeptide quantitation reagent
• EZQ phosphopeptide destain reagent
• EZQ 96-well microplate cassette
• Assay paper
• Positive control phosphopeptide pT1721
• Kemptide, a negative control peptide
• A detailed protocol for phosphopeptide quantitation (EZQ(R) Phosphopeptide Quantitation Kit)

Sufficient materials are provided for 1000 microplate-well assays. The EZQ Phosphopeptide Quantitation Kit is designed for high-throughput analysis. The solid-phase format and special 96-well microplate can be used with readily available fluorescence-based detection instruments, including either top- or bottom-reading microplate readers and laser scanners equipped with 532–560 nm lasers, as well as UV illuminators in combination with photographic or CCD cameras for image documentation and analysis (with somewhat lower sensitivity).

Pro-Q Diamond LC Phosphopeptide Detection Kit

The Pro-Q Diamond LC Phosphopeptide Detection Kit (P33203) provides sensitive and selective fluorescence-based detection of phosphorylated peptides during liquid chromatography separations. The Pro-Q Diamond LC phosphopeptide detection reagent interacts selectively with phosphoserine-, phosphothreonine- and phosphotyrosine-containing peptides to form unique, highly fluorescent dye–phosphopeptide complexes that elute from an HPLC column with altered retention times, allowing identification and purification of phosphopeptides prior to analysis by mass spectrometry. This kit is ideal for isolating phosphopeptides from chromatographic fractions of semi-complex peptide mixtures or from complex peptide mixtures such as the tryptic digest of a phosphoprotein. The Pro-Q Diamond LC Phosphopeptide Detection Kit provides:

• Pro-Q Diamond LC phosphopeptide detection reagent
• Concentrated activation buffer
• Positive control phosphopeptide RII
• Kemptide, a negative control peptide
• A detailed protocol (Pro-Q(R) Diamond LC Phosphopeptide Detection Kit)

Sufficient reagents are provided for 20 HPLC separations; a single separation will selectively detect 20 picomoles or less of a monophosphorylated peptide using a standard microbore C₁₈ HPLC column.

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.

MBDS: A Fluorogenic Reagent for Serum Albumins

4-Amino-4'-benzamidostilbene-2,2'-disulfonic acid (MBDS, A11760) is a reagent with properties similar to a commonly used probe for hydrophobic sites in proteins, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS, A47; Other Nonpolar and Amphiphilic Probes - Section 13.5; Figure 9.15). Like 1,8-ANS (Monitoring Protein Folding Processes with Anilinonaphthalenesulfonate Dyes - Note 13.3), MBDS is virtually nonfluorescent in water (quantum yield <0.01); however, upon binding to the hydrophobic pocket of serum albumins and some other proteins, it undergoes an almost 100-fold increase in its fluorescence.

Figure 9.15 Fluorescence enhancement of 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid, A47) upon binding to protein. The image shows aqueous solutions of 1,8-ANS excited by ultraviolet light. The addition of protein (bovine serum albumin) to the solution in the cuvette on the left results in intense blue fluorescence. In comparison, the fluorescence of uncomplexed free dye in the cuvette on the right is negligible.

Anti-Dinitrophenyl Antibody: A Reagent for Measuring Protein Carbonyls

Oxidative injury can be monitored by following the formation of protein-derived aldehydes and ketones. Traditionally, protein-derived aldehydes and ketones have been quantitated using a colorimetric assay based on their reaction with 2,4-dinitrophenylhydrazine to yield protein-bound dinitrophenyl moieties (DNP). A much more sensitive ELISA method has been developed that detects the protein-bound DNP using unlabeled or biotin-labeled anti-DNP antibodies (A6430, A6435; Anti-Dye and Anti-Hapten Antibodies - Section 7.4). The bound anti-DNP antibody is subsequently detected with horseradish peroxidase–conjugated secondary detection reagents (Secondary Immunoreagents - Section 7.2). Our Alexa Fluor 488 and fluorescein conjugates of the anti-DNP antibody (A11097, A6423; Secondary Immunoreagents - Section 7.2) may potentially also be applied to this detection scheme. Our polyclonal antibody to nitrotyrosine (A21285, Anti-Dye and Anti-Hapten Antibodies - Section 7.4) can be used similarly to separate and detect proteins of cell extracts that have been naturally nitrated by nitric oxide (Probes for Nitric Oxide Research - Section 18.3, Figure 18.23). Use of these rabbit polyclonal antibodies in combination with Captivate ferrofluid goat anti–rabbit IgG antibody (C21474) permits selective isolation of modified proteins and their targets from solutions (Figure 7.55, Figure 7.104).

Figure 18.23 Specificity of our rabbit anti-nitrotyrosine antibody (A21285) to nitrated proteins. Equal amounts of avidin (A887, lane 1) and CaptAvidin biotin-binding protein (C21385, lane 2) were run on an SDS-polyacrylamide gel (4–20%) and blotted onto a PVDF membrane. CaptAvidin biotin-binding protein, a derivative of avidin, has nitrated tyrosine residues in the biotin-binding site. Using the Pro-Q Western Blot Stain Kit #3 (P21864), nitrated proteins were identified with the anti-nitrotyrosine antibody, in combination with an alkaline phosphatase conjugate of goat anti–rabbit IgG antibody (G21079) and the red-fluorescent substrate, DDAO phosphate (D6487).

Figure 7.55 Flow chart for the magnetic separation and analysis of a cell suspension. Cells are treated with an antibody or a biotinylated or DSB-X biotin–labeled probe that binds to cell-surface markers. The treated cells are incubated with the appropriate Captivate ferrofluid conjugates, which bind to target cells. The mixture is then transferred to a chamber that is inserted into a magnetic yoke. Under the influence of a strong magnetic field, the cells bound to Captivate ferrofluid conjugates are rapidly separated from the unbound cells. The separate cell populations can be analyzed by both fluorometry and fluorescence microscopy.

Figure 7.104 Cell separation using Captivate ferrofluid streptavidin and DSB-X biotin conjugates. A mixed population of cells is first mixed with a DSB-X biotin–labeled antibody against an appropriate surface antigen (panel A); subsequent incubation results in the labeling of a specific subpopulation (panel B). The sample is then incubated with Captivate ferrofluid streptavidin (C21476), which binds to the DSB-X biotin hapten, allowing the labeled cells to be isolated via a magnetic field (panel C). After the unlabeled cells have been washed away, the captured cells can be released by reversing the streptavidin linkage to DSB-X biotin with unlabeled biotin (panel D).

EnzChek and Amplex Red Assay Kits

Molecular Probes prepares numerous chromogenic and fluorogenic substrates that are useful for quantitating enzymes and enzymatic activity in experimental samples. In addition, we have developed several EnzChek Assay Kits, DQ Assay Kits and Amplex Red Assay Kits especially designed for detecting a wide variety of enzymes and their substrates. Most of these products are described in Enzyme Substrates - Chapter 10.

Glutathione Agarose and Anti–Glutathione S-Transferase Antibody for GST Fusion Protein Identification and Purification

In protein fusion techniques, the coding sequence of one protein is fused in-frame with another so that the expressed hybrid protein possesses desirable properties of both parent proteins. One common partner in these engineered products is glutathione S-transferase (GST), a protein with natural binding specificity that can be exploited to facilitate its purification. Because the GST portion of the fusion protein retains its affinity and selectivity for glutathione, the fusion protein can be conveniently purified from the cell lysate in a single step by affinity chromatography on glutathione agarose (Figure 9.16). For purification of GST fusion proteins, Molecular Probes offers glutathione linked via the sulfur atom to crosslinked beaded agarose (10 mL of sedimented bead suspension, G2879, Glutathione Agarose, Linked through Sulfur). This reagent is also available from Molecular Probes in bulk quantities (100 mL of sedimented bead suspension, G21800). Each milliliter of gel can bind approximately 5–6 mg of bovine-liver GST. Adding excess free glutathione liberates the GST fragment from the matrix, which can then be regenerated by washing with a high-salt buffer.

Molecular Probes also offers a highly purified rabbit polyclonal anti-GST antibody (A5800, Labeled and Unlabeled Anti-Glutathione S-Transferase Antibodies) that can be used to purify GST fusion proteins by immunoprecipitation. This highly specific antibody, which was generated against a 260–amino acid N-terminal fragment of the Schistosoma japonica enzyme expressed in Escherichia coli, is also useful for detecting GST fusion proteins on Western blots (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4) and for detecting GST distribution in cells (Primary Antibodies for Diverse Applications - Section 7.5). The intensely green-fluorescent Alexa Fluor 488 conjugate of anti–glutathione S-transferase (A11131) is also available for direct detection of GST fusion proteins.

Our Glutathione Transferase Fusion Protein Purification Kit (G21801) facilitates isolation and characterization of GST fusion proteins. This kit, which contains sufficient materials for five isolations, contains:

• Glutathione agarose
• Anti–glutathione S-transferase antibody
• Purification columns
• A detailed protocol (Product Information Sheet)

Following purification, the fusion protein can serve as an immunogen for antibody production or its properties can be compared with those of the native polypeptide to provide insights on the normal function of the polypeptide of interest. Such methods have been used to investigate biological properties of many proteins. Examples include cleavage of the capsid assembly protein ICP35 by the herpes simplex virus type 1 protease, the role of the Rho GTP-binding protein in lbc oncogene function and the association of v-Src with cortactin in Rous sarcoma virus–transformed cells. In fact, the Ca²⁺-binding properties of a protein kinase C–GST fusion protein were examined while the GST fusion protein was still bound to the glutathione agarose. Likewise, interactions of a DNA-binding protein–GST fusion protein have been assessed using an affinity column consisting of the fusion protein bound to glutathione agarose. Alternatively, the GST fusion expression vector can be engineered to encode a recognition sequence for a site-specific protease, such as thrombin or factor Xa, between the GST structural gene and gene of interest. Once the fusion protein is bound to the affinity matrix, the site-specific enzyme can be added to release the protein.

Figure 9.16 Coomassie brilliant blue–stained SDS-polyacrylamide gel, demonstrating the purification of a glutathione S-transferase (GST) fusion protein using glutathione agarose (G2879, G21800). Lane 1 contains crude supernatant from an Escherichia coli lysate and lane 2 contains the affinity-purified GST fusion protein.

Streptavidin Agarose and CaptAvidin Agarose

Molecular Probes prepares both streptavidin and CaptAvidin biotin-binding protein conjugated to 4% beaded crosslinked agarose (S951, C21386) — matrices that can be used to isolate biotinylated peptides, proteins, hybridization probes, haptens and other molecules. In addition, biotinylated antibodies can be bound to streptavidin agarose or CaptAvidin agarose to generate affinity matrices for the large-scale isolation of antigens. For instance, streptavidin agarose has been used to isolate acetylcholine receptors from cultured myotubules after labeling the receptors with biotinylated α-bungarotoxin (B1196, Probes for Neurotransmitter Receptors - Section 16.2). Streptavidin agarose has also been used to investigate the turnover of cell-surface proteins that had previously been derivatized with an amine-reactive biotin (B1582, Biotinylation and Haptenylation Reagents - Section 4.2). The binding capacity of our streptavidin agarose is measured in an assay using fluorescein biotin (B1370, Biotin and Desthiobiotin Conjugates - Section 4.3, ). Typically, the conjugate binds 15–20 µg (18–24 nanomoles) of fluorescein biotin per milliliter of sedimented gel. Our DSB-X biotin technology (see below) makes the capture and release of antigens and receptors from solutions even easier.

CaptAvidin agarose has been specially designed to allow easier dissociation of the avidin–biotin complex (Figure 7.96). Avidin and biotin form a very strong noncovalent bond with a K_a of ~10¹⁵ M^-1. Although this high affinity is advantageous for many histochemical applications, it is a major drawback for affinity chromatography. The conditions needed to dissociate the avidin–biotin complex (8 M guanidine hydrochloride, pH 1.5) are usually too harsh for proteins and prevent the use of avidin for purifying biotinylated molecules. To address this problem, the tyrosine residues in the four biotin-binding sites of CaptAvidin biotin-binding protein (C21385, CaptAvidin Biotin-Binding Protein) are nitrated, considerably reducing its affinity for biotinylated molecules above pH 9. At pH 4.0, CaptAvidin biotin-binding protein binds biotin tightly, with a K_a of 10⁹ M^-1. At pH 10, however, this association is reversed, allowing complete dissociation of the avidin–biotin complex.

Figure 7.96 Diagram of the use of CaptAvidin agarose in affinity chromatography. A biotinylated IgG molecule and target antigen are used as an example.

Researchers have used CaptAvidin agarose affinity chromatography to purify immunoglobulins from whole rabbit serum and to isolate anti-transferrin antibody directly from rabbit IgG fractions. CaptAvidin agarose can be used to isolate cellular proteins that are selectively biotinylated with the reagents in our FluoReporter Cell-Surface Biotinylation Kit (F20650, Biotinylation and Haptenylation Reagents - Section 4.2) and to selectively isolate glycoproteins bound to the biotin-XX conjugate of concanavalin A (C21420, Lectins and Other Carbohydrate-Binding Proteins - Section 7.7). The biotin-binding capacity of CaptAvidin derivatives is at least 10 µg of biotin per mg protein.

Streptavidin Acrylamide, CaptAvidin Acrylamide and Reactive Acrylamide Derivatives

Streptavidin acrylamide (S21379, Agarose and Acrylamide Conjugates), which is prepared from the succinimidyl ester of 6-((acryloyl)amino)hexanoic acid (acryloyl-X, SE, A20770), may be useful for the preparation of biosensors. A similar streptavidin acrylamide has been shown to copolymerize with acrylamide on a polymeric surface to create a uniform monolayer of the immobilized protein. The streptavidin can then bind biotinylated ligands, including biotinylated hybridization probes, enzymes, antibodies and drugs. CaptAvidin acrylamide (C21387, CaptAvidin Biotin-Binding Protein) is expected to have similar utility, but offers an advantage — the bond that it forms with biotinylated probes is reversible at about pH 10.

Like streptavidin and CaptAvidin biotin-binding protein, other amine-containing biomolecules can be crosslinked to acrylamides using acryloyl-X, SE. Acryloyl-X, SE reacts with amines of proteins, amine-modified nucleic acids and other biomolecules to yield acrylamides that can be copolymerized into polyacrylamide matrices or on surfaces, such as in microarrays and in biosensors.

DSB-X Biotin: Easily Reversible Binding to Streptavidin Agarose

Our exclusive DSB-X biotin technology, which is described in greater detail in Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, permits the selective binding and release of proteins that are labeled with DSB-X biotin succinimidyl ester, a component of our DSB-X Biotin Protein Labeling Kit (D20655, Kits for Labeling Proteins and Nucleic Acids - Section 1.2). Temporary immobilization of a DSB-X biotin–conjugated macromolecule, such as an antibody, on streptavidin agarose permits the antibody to selectively capture antigens from solutions (Figure 7.100). Following gentle elution at neutral pH with either D-biotin or D-desthiobiotin (Figure 4.1), the DSB-X biotin–conjugated protein and its targets are completely released, permitting further analysis of the released proteins (or nucleic acids) in gels or on blots.

Figure 7.100 Diagram illustrating the use of streptavidin agarose and a DSB-X biotin bioconjugate in affinity chromatography. A DSB-X biotin–labeled IgG antibody and its target antigen are used as an example.

The DSB-X Bioconjugate Isolation Kit #1 (D20658) provides:

• Streptavidin agarose (5 mL of a sedimented bead suspension)
  
• Solutions of D-biotin or D-desthiobiotin
  
• Purification columns
  
• A recommended protocol for binding and release of DSB-X biotin conjugates (DSB-X Bioconjugate Isolation Kit #1, with streptavidin agarose)

Figure 4.1 Comparison of the structures of D-biotin (top) and D-desthiobiotin (bottom).

DSB-X biotin–labeled proteins can be prepared with the DSB-X Biotin Protein Labeling Kit (D20655, Kits for Labeling Proteins and Nucleic Acids - Section 1.2).

Captivate Ferrofluid Conjugates

The Captivate ferrofluid conjugates of streptavidin (C21476), goat anti–mouse IgG antibody (C21473) and goat anti–rabbit IgG antibody (C21474), which have been developed in cooperation with Immunicon Corporation (http://www.immunicon.com/), permit the facile isolation of biotinylated, DSB-X biotin–labeled or antibody-complexed proteins — including antibodies and their haptens, as well as receptors and their receptor ligands — using magnetic separation technology (Secondary Immunoreagents - Section 7.2, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6). Cells that have been selectively separated by the Captivate ferrofluid conjugates (Figure 7.104) can be lysed and analyzed for their proteins by standard gel electrophoresis (Detection of the Total-Protein Profile in Gels, on Blots, on Microarrays and in Capillary Electrophoresis - Section 9.3) or blotting techniques (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4). The Captivate ferrofluid streptavidin conjugate can also bind biotinylated lectins and DSB-X biotin–labeled lectins for selective isolation of glycoproteins from solutions.

The Captivate ferrofluid products are unique in that they represent the only superparamagnetic particles available that allow both cell sorting and cell-based imaging to be performed simultaneously by use of the Captivate microscope-mounted magnetic yoke assembly and associated Captivate disposable sample chambers (C24701, C24700; Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3; Figure). The Captivate microscope-mounted magnetic yoke assembly includes one free set of 10 disposable sample chambers. Use of Captivate ferrofluid streptavidin in combination with biotin- or DSB-X biotin–conjugated probes permits the simultaneous isolation, visualization and counting of cells that are targets of the antibody by any researcher with access to a standard low-cost microscope with a 10× objective (). Also, when used to capture DSB-X biotin–labeled antibodies to cell-surface antigens the Captivate ferrofluid can be completely separated from the labeled cells by incubation with D-biotin (B1595, B20656; Biotinylation and Haptenylation Reagents - Section 4.2) or D-desthiobiotin (D20657, Biotinylation and Haptenylation Reagents - Section 4.2). The extremely fast capture rate and small particle size of Captivate ferrofluid means that these products should also have significant advantages over other commercially available magnetic particles in liquid-handling robotic systems.

Captivate Microscale Phosphopeptide Isolation Kit

The Captivate Microscale Phosphopeptide Isolation Kit (C33355) 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 Captivate Microscale Phosphopeptide Isolation Kit uses modified superparamagnetic particles (suspended as a ferrofluid) that bind rapidly and selectively to phosphate groups in a reversible, noncovalent manner. In conjunction with magnetic separation devices (such as the Captivate magnetic separator for six microcentrifuge tubes or the Captivate magnetic separator for 96-well microplates (C24703, C24702; Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3), these particles provide a simple and straightforward method for isolating phosphopeptides from small sample volumes. For example, 1 picomole or less of a monophosphorylated peptide can be isolated from a volume of 5 µL. Subjecting the samples to selective β-elimination/addition modification prior to mass spectroscopy analysis allows one to distinguish between phosphorylated serine, threonine and tyrosine residues. The binding capacity of the ferrofluid is approximately 1–2 picomoles of phosphate per microgram of ferrofluid. Phosphopeptide binding is highly pH dependent, with the optimum phosphopeptide capture occurring at ~pH 4. Nonspecific binding tends to increase with increasing pH, and phosphopeptide capture efficiency tends to decrease below pH 3.5.

Each Captivate Microscale Phosphopeptide Isolation Kit provides:

• Captivate ferrofluid phosphopeptide-binding reagent
• Binding/wash buffer
• Elution buffer
• Barium hydroxide and methylamine for a β-elimination reaction and alkylation reaction, respectively, to distinguish among the three different types of peptide phosphorylation
• Phosphopeptide control mixture containing four different phosphopeptides and three peptides that are not phosphorylated

The Captivate ferrofluid phosphate-binding reagent can also be used for other applications. This ferrofluid reagent selectively binds inorganic phosphate, phosphoamino acids, phosphopeptides, ATP/GTP compounds and phosphoinositol compounds.

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.

ElutaTube Microdialysis Vials

ElutaTube microdialysis vials (Fermentas, Inc.; Hanover, Maryland, USA) are easy-to-use devices that contain two panes of dialysis membrane with a molecular weight cutoff (MWCO) of 6000–8000. These single-use devices are ideal for general dialysis, buffer exchange and concentration of protein or other samples, and they are designed for sample volumes of 10–200 µL. Molecular Probes provides ElutaTube vials in convenient packs of three (E30011), as well as in the Alexa Fluor Microscale Protein Labeling Kits, which are designed for three labelings of small amounts (20–100 µg) of protein. These kits are available for labeling protein with Alexa Fluor 488 dye (A30006), Alexa Fluor 555 dye (A30007), Alexa Fluor 594 dye (A30008), Alexa Fluor 647 dye (A30009) or biotin-XX (B30010).