Invitrogen - Molecular Probes - Section 9.2 - Quantitation and Selective Purification of Proteins in Solution

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Updated: September 16, 2007

Section 9.2 — Quantitation and Selective Purification of Proteins in Solution

Several colorimetric methods have been described for quantitating proteins in solution, including the widely used Bradford and Lowry assays, as well as an assay described by Smith that uses bicinchoninic acid (BCA). However, because they rely on absorption-based measurements, these methods are inherently limited in both sensitivity and effective range. Molecular Probes has developed four unique fluorometric methods for quantitating proteins in solution — the Quant-iT Protein Assay Kit (Q33210), the NanoOrange Protein Quantitation Kit (N6666), the CBQCA Protein Quantitation Kit (C6667) and the EZQ Protein Quantitation Kit (R33200) — that outperform all existing methods (Table 9.1). We also offer several other fluorescent reagents useful for protein detection in solution.

Quant-iT Protein Assay Kit

The Quant-iT family of assay kits provides state-of-the-art reagents for sensitive and selective quantitation of protein (Table 9.1), DNA or RNA (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 (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.

NanoOrange Protein Quantitation Kit

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 (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.

CBQCA Protein Quantitation Kit

The ATTO-TAG CBQCA reagent was originally developed as a chromatographic derivatization reagent for 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 (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.

EZQ Protein Quantitation Kit

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.

Selective Protein Quantitation in Solution

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).

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; Section 13.5; Figure 9.15). Like 1,8-ANS (Product Highlight: Monitoring Protein-Folding Processes with Anilinonaphthalenesulfonate Dyes), 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.

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; Section 7.4). The bound anti-DNP antibody is subsequently detected with horseradish peroxidase–conjugated secondary detection reagents (Section 7.2). Our Alexa Fluor 488 and fluorescein conjugates of the anti-DNP antibody (A11097, A6423; Section 7.2) may potentially also be applied to this detection scheme. Our polyclonal antibody to nitrotyrosine (A21285, Section 7.4) can be used similarly to separate and detect proteins of cell extracts that have been naturally nitrated by nitric oxide (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).

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 Chapter 10.

Selective Protein Purification

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 (Section 9.4) and for detecting GST distribution in cells (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.

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, 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, Section 4.2). The binding capacity of our streptavidin agarose is measured in an assay using fluorescein biotin (B1370, 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.

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, Section 4.2) and to selectively isolate glycoproteins bound to the biotin-XX conjugate of concanavalin A (C21420, 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 —

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