Biotinylation and Haptenylation Reagents—Section 4.2

Page Contents

• Biotin and Biotinylation Reagents
• Convenient Kits for Biotinylating Proteins
• Biotin Quantitation Assay Kits
• Haptenylation Reagents
• Data Table
• Ordering Information

We are a primary manufacturer of a diverse array of biotinylation (Biotinylation and desthiobiotinylation reagents—Table 4.1) and haptenylation (Selected haptenylation reagents and their anti-hapten antibodies—Table 4.2) reagents for labeling biomolecules. Reviews of the methods that we use to prepare biotinylated and fluorescent conjugates of antibodies have been published. To make the labeling reactions particularly easy, we have developed some very useful kits for labeling proteins with biotin, DSB-X biotin, 2,4-dinitrophenyl (DNP) or a choice of several different fluorophores, as described below. Each of the protein labeling kits contains the preferred reactive dye or hapten—many of which have spacers to reduce interactions between the label and the biomolecule—along with a detailed protocol for preparing the conjugates. In most cases, these kits also provide the separation media for purifying labeled protein conjugates from the reaction mixture.

The primary building blocks for preparing biotinylation reagents are biotin (B1595, B20656), biotin-X and biotin-XX, where "X" represents a seven-atom aminohexanoyl spacer between biotin and the reactive carboxylic acid. This spacer helps to separate the biotin moiety from its point of attachment, potentially reducing the interaction of biotin with the biomolecule to which it is conjugated and enhancing its ability to bind to the relatively deep biotin-binding sites in avidin (Figure 4.2.1). D-Desthiobiotin is the biological precursor to D-biotin and a key reagent in our DSB-X biotin technology.

We also offer biocytin (ε-biotinoyl-L-lysine, B1592), which contains a primary amine that allows it to be fixed in cells with aldehyde-based fixatives, facilitating subsequent detection with conjugates of avidin and streptavidin (Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). Biocytin derivatives, including probes that contain both biotin and fluorophore moieties, are commonly employed as microinjectable cell tracers and are discussed in Biotin and Desthiobiotin Conjugates—Section 4.3 and Polar Tracers—Section 14.3.

Figure 4.2.1 ELISA-type assay comparing the binding capacity of bovine serum albumin (BSA) and goat anti–mouse IgG antibody (GAM) biotinylated with either biotin-X succinimidyl ester (B1582) or biotin-XX succinimidyl ester (B1606). The assay was developed using horseradish peroxidase (HRP) streptavidin (S911, 0.2 µg/mL) and o-phenylenediamine dihydrochloride (OPD). The moles of biotin per mole of protein were: 4.0 biotin-X/GAM (), 4.4 biotin-XX/GAM (), 6.7 biotin-X/BSA () and 6.2 biotin-XX/GAM (). Error bars on some data points have been omitted for clarity. Reprinted with permission from .

Amine-Reactive Biotinylation Reagents

Although biotin succinimidyl ester can be used to biotinylate amines in peptides, proteins and other biomolecules, we strongly recommend the biotin-X and biotin-XX succinimidyl esters (B1582, B1606). We use biotin-X succinimidyl ester or the biotin-XX derivative to prepare all Molecular Probes biotinylated protein and dextran conjugates. Red blood cells that were biotinylated with biotin-X succinimidyl ester—but surprisingly not those modified with biotin-X sulfosuccinimidyl ester—could be detected in circulation for almost 100 days after injection into dogs.

The sulfosuccinimidyl esters of biotin-X and biotin-XX have been extensively used as topological probes to label proteins in the outer membrane surface, yielding conjugates that can be separated by electrophoresis, blotted onto membranes and then detected with a fluorophore- or enzyme-conjugated avidin derivative (Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). We utilize biotin-XX sulfosuccinimidyl ester as a component in our FluoReporter Cell-Surface Biotinylation Kit (F20650, see below).

Amine-Reactive Chromophoric Biotin Derivative

Determining a protein's degree of biotinylation is relatively difficult because of the lack of visible absorbance by the biotin molecule. To facilitate this determination, we offer an amine-reactive chromophoric derivative, biotin-X 2,4-dinitrophenyl-X-L-lysine succinimidyl ester (DNP-X–biocytin-X, SE). Following protein conjugation, the extent of biotinylation is easily determined from the absorbance of the DNP chromophore (EC_{360 nm} = 15,000 cm^-1M^-1). Incorporation of the DNP moiety into the biotinylating reagent does not affect its complexation with avidin or with anti-biotin antibodies. 

Thiol-Reactive Biotinylation Reagents

Although amine-reactive reagents are more commonly employed, the thiol-reactive biotin iodoacetamide, frequently identified in the literature by the acronym BIAM, biotin maleimide and DSB-X biotin C₂-iodoacetamide derivatives can also be used to label proteins and thiol-modified oligonucleotides. Biotin iodoacetamide and biotin maleimide are primarily used for biotinylation of free protein thiols in relation to investigations of thiol–disulfide exchange, disulfide isomerization, S-glutathionylation and other post-translational modification processes.

The TS-Link reagents are water-soluble, biotin or BODIPY thiosulfates that react readily and selectively with free thiols to form disulfide bonds (Figure 4.2.2, contact Custom Services for more information). In contrast to the thioether bonds formed by maleimides and iodoacetamides, the disulfide bond formed by this TS-Link reagent is reversible—the TS-Link biotin or BODIPY label can easily be removed using a reducing agent such as dithiothreitol or tris-(2-carboxyethyl)phosphine (DTT, D1532; TCEP, T2556; Introduction to Thiol Modification and Detection—Section 2.1), leaving the molecule of interest unchanged for downstream processing. These TS-Link reagents yield the same disulfide products as methanethiosulfonates (MTS reagents), but they are much more polar and water soluble and may therefore selectively react with residues on the surface of a protein or live cell.

Figure 4.2.2 Reaction of a TS-Link reagent (R1) with a thiol (R2), followed by removal of the label with a reducing agent.

Click-iT Biotin Alkyne and Biotin Azide Reagents

Click-iT labeling and detection technology uses bioorthogonal reactive chemistry in which the reaction partners have no endogenous representation in biological molecules, cells, tissues or model organisms. The click reaction comprises a copper-catalyzed cycloaddition between an alkyne and an azide, forming a stable triazole conjugate (Click Chemistry—Section 3.1). The azide and alkyne moieties can be used interchangeably; either one can be used to tag the biomolecule of interest, whereas the other is used for subsequent detection. Moreover, the click chemistry labels—either the alkyne or the azide—provide a functional group that typically neither reacts with other cell components nor disrupts normal cell processes. The relative transparency to cell machinery of these labels means that tagged molecules are often acceptable substrates for enzymes that assemble these building blocks into biopolymers.

Biotin alkyne (B10185) and biotin azide (B10184) are available for use in the detection and affinity capture of azide- and alkyne-modified biomolecules, respectively, using click chemistry. The biotin conjugate formed by the click reaction can subsequently be detected with a labeled avidin or streptavidin (Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). We also offer a Click-iT Biotin Protein Analysis Detection Kit (C33372, Detecting Protein Modifications—Section 9.4) for detection of azide-functionalized glycoproteins in 1D or 2D electrophoresis gels or western blots.

Histidine, Serine and Threonine Modification with Biotin Derivatives

Tripeptide sequences of certain peptides such as gonadotropin releasing hormone (GnRH), wherein serine, threonine or tyrosine residues are separated from a histidine residue by a single amino acid, can be selectively acylated by the succinimidyl ester of biotin-X (B1582). This reaction probably involves formation of an acyl histidine intermediate, followed by intramolecular transfer of the label (Figure 4.2.3). O-acylation can be detected by treating the conjugate with hydroxylamine, which cleaves esters of biotin but not amides. N-terminal serine and threonine residues of proteins can be oxidized by periodate and then biotinylated with biocytin hydrazide (B1603), which are described below.

Figure 4.2.3 Nucleophilic attack of serine on the carbonyl group (C=O) of biotin-X, SSE results in the stable O-acylated derivative. In addition to histidine-x-serine, this stable intermediate can be formed in the presence of linear sequences of histidine-x-tyrosine and histidine-x-threonine, where "x" refers to any amino acid.

Aldehyde Modification with Biotin Hydrazides and Biotin Hydroxylamine (ARP)

As described in Reagents for Modifying Aldehydes and Ketones—Section 3.3, aldehydes generated by periodate oxidation of vicinal diols in glycoproteins, polysaccharides and RNA or of N-terminal serine and threonine residues in proteins can be biotinylated using biotin-XX hydrazide. In cases where structural integrity may be compromised by periodate oxidation, derivatization with biotin hydrazide via reductive amination at the reducing end provides an alternative method for biotinylating carbohydrates. Biocytin hydrazide (B1603) may be preferred over biotin-XX hydrazide in some labeling protocols because of its higher water solubility. Biotin hydrazides are also often used for detection and affinity capture of oxidatively damaged proteins via coupling to carbonyl groups.

The biotin-containing hydroxylamine derivative ARP (aldehyde-reactive probe, A10550) has been used to modify the exposed aldehyde group at abasic lesions in DNA (Figure 4.2.4). Abasic sites are generated spontaneously or can be caused by free radicals, ionizing radiation or mutagens like methyl methanesulfonate (MMS). A quick and sensitive microplate assay for abasic sites can be performed using ARP.

In addition, ARP is membrane permeant, permitting detection of abasic sites in live cells. Once the aldehyde group in an abasic site is modified by ARP and the cells are fixed and permeabilized, the resulting biotinylated DNA can be detected with fluorescent dye–, Qdot nanocrystal– or enzyme-conjugated streptavidin conjugates (Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). Likewise, ARP can be used to detect and capture 4-hydroxynonenal (HNE)–modified proteins. ARP has also been used to immobilize IgG antibodies on streptavidin-coated monolayer surfaces with their binding sites oriented toward the solution phase.

Figure 4.2.4 Aldehyde-reactive probe (ARP) used to detect DNA damage. The biotin hydroxylamine ARP (A10550) reacts with aldehyde groups formed when reactive oxygen species depurinate DNA. This reaction forms a covalent bond linking the DNA to biotin. The biotin can then be detected using fluorophore- or enzyme-linked streptavidin.

Biotinylation of Carboxylic Acids

Like other biotin amines and hydrazides, biocytin can be coupled to chemically activated carboxylic acids (Figure 4.2.5). The amine-containing biotin derivatives (B1592) are versatile intermediates for coupling biotin to DNA, carboxylic acids and array support surfaces. Biotin cadaverines are useful for transglutaminase-mediated modification of glutamine residues in cells and certain proteins (Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4, Figure 4.2.6) and for the microplate assay of transglutaminase activity.

Figure 4.2.5 Conversion of a carboxylic acid group into an aliphatic amine. The activated carboxylic acid is derivatized with a half-protected aliphatic diamine (mono-N-(t-BOC)-propylenediamine), usually in an organic solvent, followed by removal of the t-BOC–protecting group with trifluoroacetic acid.

Figure 4.2.6 Transglutaminase-mediated labeling of a protein using dansyl cadaverine.

Reactive DSB-X Biotin Derivatives

Our unique DSB-X biotin technology (Introduction to Avidin-Biotin and Antibody-Hapten Techniques—Section 4.1) permits the readily reversible binding of DSB-X biotin–labeled biomolecules to avidin and streptavidin derivatives. The DSB-X biotin reagents, which are derivatives of desthiobiotin (Figure 4.2.7), have a moderate affinity for avidins (K_d for DSB binding to streptavidin is 1.9 nM ), making DSB-X biotin an ideal ligand for transient immobilization of avidin and streptavidin conjugates. DSB-X biotin succinimidyl ester, which is a component of the DSB-X Biotin Protein Labeling Kit (D20655) described below, can be conjugated to amine-containing molecules in the same way as the biotin succinimidyl esters.

Figure 4.2.7 Comparison of the structures of D-biotin (top) and D-desthiobiotin (bottom).

APEX Biotin-XX Antibody Labeling Kit

The APEX Biotin-XX Antibody Labeling Kit (A10495, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provides a convenient method for covalently labeling small amounts (10–20 µg) of IgG antibody with biotin-XX sulfosuccinimidyl ester (biotin-XX SSE). The water-soluble biotin-XX SSE has a 14-atom spacer that enhances the binding of biotin derivatives to avidin's relatively deep binding sites. APEX Antibody Labeling Kits are specifically designed to allow labeling of small amounts of IgG antibody, even in the presence of contaminants. These kits utilize a solid-phase labeling technique that captures the IgG antibody on resin inside an APEX antibody labeling tip (Figure 4.2.8). Any contaminants, including stabilizing proteins or amine-containing buffers, are eluted through the tip before labeling. After applying the amine-reactive biotin to the IgG antibody trapped on the resin, a biotinylated IgG conjugate is formed and subsequently eluted from the resin using elution buffer. The biotinylated IgG conjugate is ready to use in an imaging or flow cytometry assay in as little as 2.5 hours with minimal hands-on time. The typical yield of labeled antibody using this method is between 40 and 80%.

Each APEX Biotin-XX Antibody Labeling Kit provides all reagents required to perform five separate labeling reactions of 10–20 µg IgG antibody, including:

• Five vials of amine-reactive biotin-XX sulfosuccinimidyl ester (biotin-XX SSE)
• Five APEX antibody labeling tips
• Wash, labeling, neutralization and elution buffers
• Dimethylsulfoxide (DMSO)
• Labeling protocol (APEX Antibody Labeling Kits)

For labeling larger amounts of protein, we recommend the Biotin-XX Microscale Protein Labeling Kit, which is optimized for 20–100 µg samples of proteins between 10,000 and 150,000 daltons; the FluoReporter Mini-Biotin-XX Protein Labeling Kit, which is optimized for 0.1–3 mg samples of >30,000-dalton proteins; or the FluoReporter Biotin-XX Protein Labeling Kit, which is optimized for 5–20 mg samples.

Figure 4.2.8 Illustration of the use of the APEX antibody labeling tip, provided in APEX Antibody Labeling Kits. A) Applying solutions to the resin in the tip. B) Pushing solutions onto the resin in the tip by attaching the APEX antibody labeling tip to a pipette.

Biotin-XX Microscale Protein Labeling Kit

The Biotin-XX Microscale Protein Labeling Kit (B30010, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provides a convenient means for biotinylating small amounts (20–100 µg) of purified protein. The kit has been optimized for labeling proteins with molecular weights between 12,000 and 150,000 daltons, and contains everything needed to perform three labeling reactions and to separate the resulting conjugates from excess reactive biotin. Convenient spin columns are used to purify the labeled protein with yields between 60 and 90%, depending primarily on the molecular weight of the starting material. Labeling and purification can be completed in as little as 30 minutes.

Each Biotin-XX Microscale Protein Labeling Kit contains:

• Biotin-XX sulfosuccinimidyl ester
• Sodium bicarbonate
• Reaction tubes
• Purification resin and spin filters
• Protocols for preparing and purifying the biotinylated protein (Biotin-XX Microscale Protein Labeling Kit) 

For determining the degree of labeling, the FluoReporter Biotin Quantitation Assay Kit for proteins is available separately (F30751) or in combination with the Biotin-XX Microscale Protein Labeling Kit (B30756). When biotinylating larger amounts of protein, we recommend the FluoReporter Mini-Biotin-XX Protein Labeling Kit, which is optimized for 0.1–3 mg samples of >40,000-dalton proteins, or the FluoReporter Biotin-XX Protein Labeling Kit, which is optimized for 5–20 mg samples; see below for a description of these kits.

FluoReporter Mini-Biotin-XX Protein Labeling Kit

The FluoReporter Mini-Biotin-XX Protein Labeling Kit (F6347, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provides a method for efficiently biotinylating small amounts of antibodies or other proteins. The water-soluble biotin-XX sulfosuccinimidyl ester contained in this kit readily reacts with a protein's amines to yield a biotin moiety that is linked to the protein through two tandem aminohexanoyl chains ("XX"). This 14-atom spacer has been shown to enhance the binding of biotin derivatives to avidin's relatively deep binding sites (Figure 4.2.1).

Each FluoReporter Mini-Biotin-XX Labeling Kit contains:

• Biotin-XX sulfosuccinimidyl ester
• Reaction tubes, each containing a magnetic spin bar
• Purification resin, spin columns and collection tubes
• Dialysis tubing (molecular weight cut-off ~12,000–14,000 daltons)
• Protocols for preparing and purifying the biotinylated protein
  

The ready-to-use spin columns provide an extremely convenient method of purifying the biotinylated protein from excess biotinylation reagents. Alternatively, the researcher may choose to remove excess reagents by dialysis, thereby avoiding further dilution of the biotinylated protein. The FluoReporter Mini-Biotin-XX Protein Labeling Kit contains sufficient reagents for five biotinylation reactions of 0.1–3 mg of protein each.

DSB-X Biotin Protein Labeling Kit

Our unique DSB-X biotin technology permits the facile reversal of the typically irreversible biotin–avidin interaction under extremely gentle conditions. DSB-X biotin succinimidyl ester, a derivative of desthiobiotin (Figure 4.2.7) with an additional seven-atom spacer, reacts with amine groups of biomolecules to form stable amides. The DSB-X biotin conjugate can be detected with any of the avidin or streptavidin derivatives described in Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9. Binding is almost totally reversed by addition of free biotin (B1595, B20656) at neutral pH and normal ionic strength. Significantly, DSB-X biotin–conjugated biopolymers can be separated from complex mixtures using agarose affinity matrices.

The DSB-X Biotin Protein Labeling Kit (D20655, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) contains the reagents required for five protein conjugations of 0.5–3 mg each, including:

• DSB-X biotin succinimidyl ester
• Dimethylsulfoxide (DMSO)
• Reaction tubes
• Purification resin, spin columns and collection tubes for small-scale purifications
• Dialysis tubing for larger-scale purifications
• Detailed protocols for conjugations and purifications (DSB-X Biotin Protein Labeling Kit)

FluoReporter Cell-Surface Biotinylation Kit

Biotin-XX sulfosuccinimidyl ester is a membrane-impermeant, amine-reactive compound that may be used to label proteins exposed on the surface of live cells (Figure 4.2.9). The sulfosuccinimidyl ester forms an extremely stable conjugate with cell-surface proteins, and the biotin provides a convenient hapten for subsequent isolation or analysis with an avidin-based protein, including streptavidin, NeutrAvidin and CaptAvidin biotin-binding proteins (Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). Cell-surface biotinylation techniques have been employed to differentially label proteins in the apical and basolateral plasma membranes of epithelial cells. These techniques are also well suited for studying internalization of membrane proteins and cell-surface targeting of proteins.

The FluoReporter Cell-Surface Biotinylation Kit (F20650) provides a convenient method to label proteins exposed on the cell surface including, but not limited to, membrane proteins. This kit includes:

• Biotin-XX sulfosuccinimidyl ester
• Anhydrous DMSO for preparing stable stock solutions
• Detailed protocols for cell-surface biotinylation (FluoReporter Cell-Surface Biotinylation Kit)

The supplied protocol for cell-surface biotinylation is easy to perform and can be completed in less than one hour. Biotinylated proteins can be subsequently identified using western blot techniques and labeled avidin conjugates described in Qdot Nanocrystals—Section 6.6 and Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6.

Figure 4.2.9 Identification of cell-surface proteins in Jurkat cells labeled with the FluoReporter Cell-Surface Biotinylation Kit (F20650). The labeled cells were fractionated by differential detergent extraction into membrane and cytosolic fractions. The proteins were then precipitated with acetone, separated on an SDS-polyacrylamide gel and blotted onto a PVDF membrane. Total proteins were detected with the SYPRO Ruby protein blot stain component of the kit (left panel); biotinylated proteins were identified with alkaline phosphatase streptavidin in combination with the red-fluorescent substrate, DDAO phosphate (right panel). MW = protein molecular weight markers; Con A = biotinylated concanavalin A.

Zenon Biotin Antibody Labeling Kits

Zenon Antibody Labeling Kits provide a fast, versatile and reliable method for producing antibody conjugates that can be used in any application where a directly labeled primary antibody is suitable, including microscopy, flow cytometry, high-throughput screening, and others. This enabling technology not only eliminates the need for secondary detection reagents in many protocols, but also simplifies immunolabeling protocols that previously were time consuming or impractical, such as use of multiple antibodies derived from the same species in the same protocol. We have applied our exclusive Zenon technology (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3) in several  Zenon Biotin-XX Antibody Labeling Kits, which permit the quantitative biotinylation of even submicrogram quantities of an antibody in less than 10 minutes.

The Zenon Biotin-XX Antibody Labeling Kits include:

• Zenon Biotin-XX Mouse IgG₁ Labeling Kit (Z25052)
• Zenon Biotin XX Mouse IgG_2a Labeling Kit (Z25152)
• Zenon Biotin-XX Rabbit IgG Labeling Kit (Z25352)
• Zenon Biotin-XX Human IgG Labeling Kit (Z25452)

Zenon Antibody Labeling Kits are designed to label intact primary antibodies in amounts from less than 1 µg to as much as 50 µg, starting with a purified antibody fraction or with a crude antibody preparation such as ascites fluid or a hybridoma supernatant. Zenon labeling technology takes advantage of the immunoselectivity of the antibody binding reaction by forming a complex between an intact primary IgG antibody and a biotin-labeled Fab fragment directed against the Fc portion of the IgG. Simple mixing of the labeled Fab fragment supplied in the Zenon Antibody Labeling Kit with the corresponding primary antibody quantitatively produces  the Fab–antibody complex in under 10 minutes, with no pre- or post-labeling purification required (Figure 4.2.10). This labeled Fab–antibody complex can be immediately used to stain cells, tissues and other targets in the same manner as a covalently labeled primary antibody.

Each Zenon Biotin Antibody Labeling Kit provides:

• Zenon antibody labeling reagent
• Zenon blocking reagent
• Detailed protocols for antibody labeling (Zenon Mouse IgG Labeling Kits)

The Zenon Biotin Antibody Labeling Kit contains sufficient reagents for 50 labelings, where one labeling is defined as the amount of Zenon labeling reagent required to label 1 µg of an intact, affinity-purified IgG antibody at a Fab:antibody molar ratio of 3:1.

Figure 4.2.10 Labeling scheme utilized in the Zenon Antibody Labeling Kits. A) An unlabeled IgG antibody is incubated with the Zenon labeling reagent, which contains a fluorophore-labeled, Fc-specific anti-IgG Fab fragment. B) This labeled Fab fragment binds to the Fc portion of the IgG antibody. C) Excess Fab fragment is then neutralized by the addition of a nonspecific IgG, preventing crosslabeling by the Fab fragment in experiments where primary antibodies of the same type are present. Note that the Fab fragment used for labeling need not be coupled to a fluorophore, but could instead be coupled to an enzyme (such as HRP) or to biotin.

FluoReporter Biotin Quantitation Assay Kit for Biotinylated Proteins

The FluoReporter Biotin Quantitation Assay Kit for biotinylated proteins (F30751) provides a sensitive fluorometric assay for determining the number of biotin labels on a protein. The assay is based on the displacement of a ligand tagged with a quencher dye from the biotin-binding sites of Biotective Green reagent (Figure 4.2.11). The assay can detect from 4 to 80 picomoles of biotin in a sample (Figure 4.2.12), providing a 50-fold higher sensitivity than the traditional HABA biotin-binding assay. Furthermore, unlike the HABA biotin-binding assay, which requires ~1 mg of protein sample, the FluoReporter biotin quantitation assay requires only a minimum of 600 ng of a singly biotinylated IgG of MW 150,000. For proteins of lower molecular weight or multiple biotin labels, less protein can be used. To expose any biotin groups in a multiply labeled protein that are sterically restricted and inaccessible to the Biotective Green reagent, this kit includes protease and an optional protocol for digesting the protein. After this preliminary digestion, biotin assay values agree well with MALDI-TOF determinations. With excitation/emission maxima of 495/519 nm, this assay is compatible with any fluorescence-based microplate reader capable of detecting fluorescein (FITC) or Alexa Fluor 488 dye; it can also be scaled up for fluorometer-based experiments.

Each FluoReporter Biotin Quantitation Assay Kit for biotinylated proteins includes:

• Biotective Green reagent
• Biocytin
• Protease
• Concentrated phosphate-buffered saline (PBS)
• Biotinylated goat anti–mouse IgG antibody
• Detailed protocols (FluoReporter Biotin Quantitation Assay Kit For Biotinylated Proteins) 

Sufficient reagents are provided for assaying 5 samples independently using eight wells in triplicate for the standard curve and three dilutions of the sample in triplicate (totaling 33 wells per assay). However, fewer wells may be used to conserve sample and a single standard curve can be used for multiple samples in the same experimental session. Biocytin (biotinylated lysine) is provided as a standard for the assay because it more closely represents the form of biotin present after proteolytic cleavage. Biotinylated goat anti–mouse IgG antibody is also provided as a positive control and biotinylated protein standard.

Figure 4.2.11 Schematic representation of the FluoReporter biotin quantitation assay. This assay uses Biotective Green reagent, which consists of avidin labeled with a fluorescent dye (D) and with quencher dye ligands (Q) occupying the biotin-binding sites. Through fluorescence resonance energy transfer (FRET), the ligand quenches the fluorescence. Biotin (B) attached to a protein displaces the quencher dye from Biotective Green reagent, yielding fluorescence proportional to the amount of added biotin.

Figure 4.2.12 Standard curve showing dynamic range of the FluoReporter Biotin Quantitation Assay Kit. Each reaction consisted of 1X PBS, 1X Biotective Green reagent and biocytin in a total volume of 100 µL. After a 5-minute incubation at room temperature in the dark, fluorescence was measured in a microplate reader using excitation at 485 ± 10 nm and fluorescence emission at 530 ± 12.5 nm.

A prerequisite for multicolor applications such as fluorescence in situ hybridization is the availability of multiple hapten molecules, along with their complementary binding proteins. The avidin–biotin system can provide only single-color detection, whereas antibody–hapten methods can generate a number of unique signals, limited only by the specificity of the antibody–hapten detection and the ability to distinguish the signals of different antibodies. The characteristics of a suitable hapten include a unique chemical structure that is not commonly found in cells (a bioorthogonal label), a high degree of antigenicity that elicits good antibody production, and a means for incorporating the hapten into the detection system. Our selection of haptenylation reagents enables researchers to covalently attach haptens to proteins, nucleotides and other biomolecules.

In addition to our wide range of biotinylation reagents discussed above, we provide many unique haptenylation reagents, including an amine-reactive version of digoxigenin (A2952), dinitrophenyl-X (DNP-X, SE; D2248) and several fluorophores (Selected haptenylation reagents and their anti-hapten antibodies—Table 4.2). We usually recommend haptenylation reagents that contain spacers between the hapten and the reactive groups to reduce potential interactions with the biomolecule to which it is conjugated and to make the hapten maximally available to secondary detection reagents. Most of the preferred haptenylation reagents in Selected haptenylation reagents and their anti-hapten antibodies—Table 4.2 possess this feature.

Fluorescein has been found to be an excellent hapten for in situ hybridization because it binds with high affinity to its anti-fluorescein antibody. Anti-fluorescein antibodies crossreact with all of the Oregon Green dyes (Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5), permitting their use with conjugates prepared from these dyes. By adding antibodies that recognize the Alexa Fluor 488, dansyl, tetramethylrhodamine and Texas Red fluorophores to our line of detection reagents (Anti-Dye and Anti-Hapten Antibodies—Section 7.4), we have greatly expanded the number of potential haptens. Because the anti-tetramethylrhodamine and anti–Texas Red dye antibodies crossreact with the tetramethylrhodamine, Lissamine rhodamine, Rhodamine Red and Texas Red fluorophores, these antibody–fluorophore combinations should not be used simultaneously to generate separate signals in a multicolor experiment. Similarly, our antibody to the BODIPY FL dye crossreacts with some of the other BODIPY dyes (BODIPY Dye Series—Section 1.4).

For a detailed explanation of column headings, see Definitions of Data Table Contents