| Updated: January 31, 2006 |
Section 4.2 — Biotinylation and Haptenylation Reagents
Molecular Probes is the primary manufacturer of a diverse array of biotinylation (Table 4.1) and haptenylation (Table 4.2) reagents for labeling biomolecules. In addition, our DSB-X biotin technology (Section 7.6) employs a modified biotin to provide a means of labeling and separating biomolecules, as well as live cells, under extremely gentle conditions.
Biotinylated molecules, as well as DSB-X biotin–labeled molecules prepared using our DSB-X Biotin Protein Labeling Kit (D20655, Section 1.2), can be subsequently detected with fluorescent dye or enzyme conjugates of avidins (Section 7.6, Table 7.23) or with NANOGOLD streptavidin gold clusters (N24918, A24925, A24926; Section 7.6). These biotinylated and DSB-X biotin–labeled molecules are also readily captured and separated from solutions with streptavidin agarose (S951, Section 7.6, Figure 7.100), CaptAvidin agarose (C21386, Section 7.6) or the streptavidin conjugate of Captivate ferrofluid superparamagnetic particles (C21476, Section 7.6). Adsorption of DSB-X biotin–labeled molecules or cells onto affinity matrices can be rapidly reversed at neutral pH and room temperature by adding free biotin to the solution (Figure 7.96).
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.
Our ARES DNA Labeling Kits (Section 8.2, Table 8.9, Figure 8.45), ULYSIS Nucleic Acid Labeling Kits (Section 8.2, Table 8.8, Figure 8.42), Alexa Fluor Oligonucleotide Amine Labeling Kits (Section 8.2, Table 8.10), ChromaTide UTP, dUTP and dCTP nucleotides (Section 8.2; Table 8.6, Table 8.7) and 5-aminohexylacrylamido-dUTP (aha-dUTP) nucleotides (Section 8.2, Table 8.7) yield probes whose labels can, in many cases, be utilized as haptens in various signal amplification schemes.
Our exclusive Zenon technology (Section 7.3) permits the quantitative biotinylation and desthiobiotinylation of even submicrogram quantities of an antibody in less than 10 minutes (Figure 7.56). The Zenon Biotin-XX and DSB-X Biotin Antibody Labeling Kits (Table 7.14) provide the reagents and detailed instructions for this labeling. Also available are numerous other Zenon Antibody Labeling Kits (Table 7.14) for labeling mouse, rabbit, goat and human IgG antibodies with many of our brightest proprietary fluorescent dyes, as well as with phycobiliproteins and enzyme labels. An outstanding feature of the Zenon technology is that it potentially permits the use of multiple antibodies derived from the same species in the same protocol.
A Variety of Biotinylation Reagents
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 4.3). We also offer biocytin (ε-biotinoyl-L-lysine, B1592), biotin ethylenediamine (A1593), biotin cadaverine (A1594), biotin-X cadaverine (B1596), DSB-X desthiobiocytin (ε-desthiobiotinoyl-L-lysine, D20652) and DSB-X biotin ethylenediamine (D30752), each of 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 (Section 7.6) or with our anti-biotin monoclonal antibody (Section 7.4). Biocytin derivatives, including probes that contain both biotin and fluorophore moieties, are commonly employed as microinjectable cell tracers and are discussed in Section 4.3 and Section 14.3. D-Desthiobiotin (D20657) is the biological precursor to D-biotin and a key reagent in our important DSB-X biotin technology (Section 7.6).
Amine-Reactive Biotinylation Reagents
Although Molecular Probes' biotin succinimidyl ester (B1513) 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), and especially the water-soluble biotin-X and biotin-XX sulfosuccinimidyl esters (B6353, B6352), because their additional 7- and 14-atom spacers greatly facilitate binding to avidin and to anti-biotin antibodies (Figure 4.3). Molecular Probes uses biotin-X succinimidyl ester or the biotin-XX derivative to prepare all of its 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 (B6353, B6352) have been extensively used as topological probes to label proteins in the outer membrane surface, yielding labeled proteins that can be separated by electrophoresis, blotted onto membranes and then detected with a fluorophore- or enzyme-conjugated avidin derivative (Section 7.6). We utilize biotin-XX sulfosuccinimidyl ester as a component in our FluoReporter Cell-Surface Biotinylation Kit (F20650, see below). Our alkaline phosphatase streptavidin–containing Pro-Q Western Blot Stain Kits (P21862, P21865; Section 9.4), Amplex Gold Western Blot Stain Kit #3 (A21892, Section 9.4) and DyeChrome Western Blot Stain Kits (D21883, D21886; Section 9.4) use biotinylated reagents to provide sensitivity similar to that of enhanced chemiluminescence but with permanent labeling.
Reactive DSB-X Biotin Derivatives
Our unique DSB-X biotin technology (Section 4.1) permits the readily reversible binding of DSB-X biotin–labeled biomolecules to avidin and streptavidin derivatives, including dye, enzyme and NANOGOLD or Alexa Fluor FluoroNanogold 1.4 nm gold cluster (N24918, A24925, A24926; Section 7.6) conjugates, affinity matrices and our Captivate ferrofluid streptavidin (C21476, Section 7.6). The DSB-X biotin reagents, which are derivatives of desthiobiotin (Figure 4.1), have a moderate affinity for avidins, making DSB-X biotin an ideal ligand for transient immobilization of avidin and streptavidin conjugates (Figure 7.106). 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. Our DSB-X biotin C2-iodoacetamide (D30753) and TS-Link DSB-X biotin C5-thiosulfate (T30754) are thiol-reactive derivatives for labeling proteins and thiol-modified oligonucleotides. In addition, we offer DSB-X biotin hydrazide (D20653), DSB-X desthiobiocytin (D20652) and DSB-X biotin ethylenediamine (D30752), reactive derivatives of DSB-X biotin that should be useful as fixable polar tracers in neurons and other cells (Section 14.3). See Section 7.6 for a complete discussion of our DSB-X biotin technology and its uses.
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, Molecular Probes offers an amine-reactive chromophoric derivative, biotin-X 2,4-dinitrophenyl-X-L-lysine succinimidyl ester (DNP-X–biocytin-X, SE; B2604; 
). Following protein conjugation, the extent of biotinylation is easily determined from the absorbance of the DNP chromophore (ε360 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. Our FluoReporter Biotin/DNP Protein Labeling Kit (F6348), described below, contains sufficient DNP-X–biocytin-X SE for 5 to 10 protein labeling reactions of 0.2–2 mg each.
The DNP-X–biocytin-X succinimidyl ester is a unique amine-reactive reagent with versatile applications. Because this reagent comprises both DNP and biotin moieties (), molecules labeled with it may be probed with Molecular Probes' anti-DNP or anti-biotin antibodies (Section 7.4, Table 7.20) or with our avidin conjugates (Section 7.6), thus facilitating correlated fluorescence and electron microscopy studies. This chromophoric biotin is also very useful for preparing conjugates for Molecular Devices' patented Threshold Immuno-Ligand Assay System. The Threshold System is designed to quantitate DNA and protein impurities in biopharmaceuticals by a urease-mediated signal amplification system that employs a silicon sensor to convert the chemical signal into an electronic signal.
Thiol-Reactive Biotinylation Reagents
Although amine-reactive reagents are more commonly employed, the thiol-reactive biotin iodoacetamide (B1591), biotin maleimide (M1602) and DSB-X biotin C2-iodoacetamide (D30753) derivatives can also be used to label proteins and thiol-modified oligonucleotides. Electrophoretically separated thiolated proteins treated with biotin maleimide have been detected in Western blots by an avidin–alkaline phosphatase conjugate. Biotin iodoacetamide was used as an enzyme substrate in an unusual chemical reaction catalyzed by a ribozyme.
We also offer TS-Link DSB-X biotin C5-thiosulfate (TS-Link desthiobiotin-X C5-thiosulfate, T30754), which is a water-soluble thiosulfate that reacts readily and selectively with a free thiol to form a disulfide bond (Figure 2.9). 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 DSB-X hapten can easily be removed using a reducing agent such as dithiothreitol (DTT, D1532; Section 2.1) or tris-(2-carboxyethyl)phosphine (TCEP, T2556; Section 2.1), leaving the molecule of interest unchanged. In addition to TS-Link DSB-X biotin C5-thiosulfate, we prepare fluorescent TS-Link BODIPY thiosulfates (Section 2.2), as well as TS-Link bimane thiosulfate (T30502, Section 2.3) and TS-Link TFP-X thiosulfate (T30875, Section 5.2).
Convenient Kits for Biotinylating Proteins
Biotin-XX Microscale Protein Labeling Kit
The Biotin-XX Microscale Protein Labeling Kit (B30010) 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
- Spin filters
- Purification resin
- ElutaTube microdialysis vials
- A protocol 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
Molecular Probes' FluoReporter Mini-Biotin-XX Protein Labeling Kit (F6347, 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 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 4.3). Each FluoReporter Mini-Biotin-XX Labeling Kit contains:
- Biotin-XX sulfosuccinimidyl ester
- Reaction tubes, each containing a magnetic spin bar
- Spin columns plus collection tubes
- Dialysis tubing (molecular weight cut-off ~12,000–14,000 daltons)
- A protocol for preparing and purifying the biotinylated protein (FluoReporter Mini-Biotin-XX Labeling Kit)
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. The Zenon Biotin-XX Antibody Labeling Kits (Table 7.14) are a useful alternative for rapid and quantitative modification of antibodies with biotin. See Section 7.3 for a complete description of our Zenon technology and Zenon Antibody Labeling Kits.
FluoReporter Biotin-XX Protein Labeling Kit
We also offer the FluoReporter Biotin-XX Protein Labeling Kit (F2610, Table 1.3) for larger-scale biotinylation reactions. Once the labeled protein is purified from excess biotin reagent, its degree of biotinylation can be determined using an avidin–biotin displacement assay; biotinylated goat IgG is provided as a control. The FluoReporter Biotin-XX Protein Labeling Kit supplies:
- Biotin-XX succinimidyl ester
- Dimethylsulfoxide (DMSO)
- Gel filtration column
- Avidin–HABA complex
- Biotinylated goat IgG
- A protocol for preparing and purifying the biotinylated protein, as well as for quantitating the degree of labeling (FluoReporter Biotin-XX Protein Labeling Kit)
The FluoReporter Biotin-XX Protein Labeling Kit provides sufficient reagents for five labeling reactions of 5–20 mg of protein each.
FluoReporter Biotin/DNP Protein Labeling Kit
The FluoReporter Biotin/DNP Protein Labeling Kit (F6348, Table 1.3) is similar to our other FluoReporter Protein Labeling Kits, except that it contains DNP-X–biocytin-X succinimidyl ester as the reactive label. When proteins are labeled with this chromophoric biotin derivative, the degree of biotinylation can be readily assessed from the extinction coefficient of DNP (ε360 nm = 15,000 cm-1M-1). An additional feature of the conjugates labeled with DNP-X–biocytin-X succinimidyl ester is that they can be recognized by avidin derivatives (or anti-biotin antibodies) and by anti-DNP antibodies, enabling researchers to choose among several detection techniques suitable for fluorescence and electron microscopy. Each FluoReporter Biotin/DNP Protein Labeling Kit contains:
- DNP-X–biocytin-X succinimidyl ester
- Dimethylsulfoxide (DMSO)
- Reaction tubes, each containing a magnetic spin bar
- Spin columns plus collection tubes
- A protocol for preparing and purifying the protein conjugate, as well as for quantitating the degree of labeling (FluoReporter Biotin/DNP Protein Labeling Kit)
The FluoReporter Biotin/DNP Protein Labeling Kit supplies sufficient reagents for 5 to 10 labeling reactions of 0.2–2 mg of protein each.
DSB-X Biotin Protein Labeling Kit
Our unique DSB-X biotin technology, which is described in detail in Section 7.6, permits the facile reversal of the virtually irreversible biotin–avidin interaction under extremely gentle conditions. DSB-X biotin succinimidyl ester, a derivative of desthiobiotin (Figure 4.1) 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 Section 7.6. Binding is almost totally reversed by addition of free biotin (B1595, B20656; Section 4.2) at neutral pH and normal ionic strength. Significantly, DSB-X biotin–conjugated biopolymers can be separated from complex mixtures using agarose affinity matrices (Figure 7.96) or our Captivate ferrofluid superparamagnetic particles (Figure 7.104). Magnetic separation can include cells targeted by the DSB-X biotin conjugate. Our DSB-X Bioconjugate Isolation Kits #1 and #2 (D20658, D20659; Section 7.6) provide the reagents and protocols for using DSB-X biotin conjugates. The DSB-X Biotin Protein Labeling Kit (D20655, 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
- A detailed protocol for conjugations and purifications (DSB-X Biotin Protein Labeling Kit)
As an alternative to direct labeling of primary antibodies with the DSB-X biotin succinimidyl ester, our Zenon DSB-X Biotin Mouse IgG1 Labeling Kit (Z25053, Section 7.3) gives rapid and quantitative complex formation with any whole mouse IgG1 monoclonal antibody.
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 4.5). 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 and the Captivate ferrofluid streptavidin conjugate (C21476, Section 7.6). 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:
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 the reagents in some of our Pro-Q, Amplex Gold and DyeChrome Western Blot Kits (Section 9.4).
Biotin Quantitation Assay Kits
FluoReporter Biotin Quantitation Assay Kit for Biotinylated Proteins
The FluoReporter Biotin Quantitation Assay Kit for biotinylated proteins (F30751) provides a sensitive fluorometric assay for accurately 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. The assay can detect from 4 to 80 picomoles of biotin in a sample, providing a 50-fold higher sensitivity than the HABA biotin binding assay described by Green. 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, the kit includes protease and an optional protocol for digesting the protein. With this preliminary digestion, biotin assay values agree well with MALDI-TOF determinations. The signal window of this assay has a Z' factor of 0.93. 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:
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.
FluoReporter Biotin Quantitation Assay Kit for Biotinylated Nucleic Acids
The FluoReporter Biotin Quantitation Assay Kit for biotinylated nucleic acids (F30755) provides a sensitive fluorometric assay for determining the number of biotin labels on a nucleic acid. The assay is based on the displacement of a quencher dye from the biotin binding sites of Biotective Green reagent. The assay can detect from 4 to 80 picomoles of biotin in a sample, providing a 50-fold higher sensitivity than the HABA biotin binding assay described by Green. Analysis of multiply biotinylated nucleic acids requires a preliminary nuclease digestion step to avoid underestimation caused by steric restriction of avidin binding. This kit is ideal for determining the degree of biotinylation of cDNA samples used in Affymetrix or RLS microarray protocols, and can be applied to as little as 13 ng of biotin-labeled nucleic acid (Table 4.3). The signal window of this assay has a Z' factor of 0.91.
Each FluoReporter Biotin Quantitation Assay Kit for biotinylated nucleic acids includes:
Sufficient reagents are provided for assaying 10 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.
Oligohistidine Fusion Protein Detection
The oligohistidine domain is a Ni2+-binding moiety comprising four to six consecutive histidine residues. Biotin-X nitrilotriacetic acid (biotin-X NTA, B11790; ) is designed to detect oligohistidine fusion proteins immobilized on nitrocellulose membranes. The nitrilotriacetic acid moiety of biotin-X NTA chelates Ni2+ that is associated with oligohistidine domains. Once bound, the complex can be detected using standard enzyme-linked streptavidin methods. McMahan and Burgess have shown that biotin-X NTA can be used to detect less than 0.1 picomole of an oligohistidine fusion protein using horseradish peroxidase streptavidin and chemiluminescence techniques. An additional benefit of biotin-X NTA is that it can be removed from the oligohistidine fusion protein at pH 4.8, allowing the blot to be reanalyzed with another probe. In addition to biotin-X NTA as a stand-alone reagent (B11790), Molecular Probes has utilized biotin-X NTA in its Pro-Q Oligohistidine Blot Stain Kits (P21878, P21879), which are discussed in detail in Section 9.4. We have also developed the Pro-Q Sapphire Oligohistidine Gel Stains (P21876, P21877; Section 9.4), which can detect oligohistidine fusion proteins without blotting (
, ). In addition, Molecular Probes offers the Penta·His mouse IgG monoclonal antibody (P21315, Section 9.4), which provides a sensitive method for specific detection of fusion proteins that have an oligohistidine domain comprising five or six consecutive histidine residues on blots and in cells.
Pro-Q Oligohistidine Blot Stain Kits
The Pro-Q Oligohistidine Blot Stain Kits (P21878, P21879; Section 9.4) include biotin-X nitrilotriacetic acid (biotin-X NTA, B11790), which chelates Ni2+ associated with oligohistidine domains. The blot is incubated with a complex of biotin-X NTA, Ni2+ and alkaline phosphatase streptavidin. The complex binds to the oligohistidine domain and is then detected by reaction with a fluorogenic phosphatase substrate, either DDAO phosphate (Kit #1, P21878) or ELF 39 phosphate (Kit #2, P21879). The enzymatic reactions produce fluorescent products within minutes. Both fluorogenic detection methods have sensitivity rivaling that of ECL detection, but the signals can be imaged several times and are stable indefinitely on dried blots. The green-fluorescent ELF 39 product (Kit #2) forms a permanent precipitate, whereas the red-fluorescent DDAO (Kit #1) can be washed off for further processing of the blot.
Pro-Q Sapphire Oligohistidine Gel Stains
The Pro-Q Sapphire 365 oligohistidine stain and Pro-Q Sapphire 488 oligohistidine stain (P21876, P21877; Section 9.4) use proprietary reagents that allow detection of oligohistidine fusion proteins directly in the gel (), eliminating the need to blot the proteins to a membrane for immunodetection. Both reagents use one of Molecular Probes' state-of-the-art fluorescent dyes conjugated to a nitrilotriacetic acid (NTA) moiety. The NTA moiety binds to oligohistidine domains, providing a simple and highly specific detection method. As little as 50 ng of oligohistidine fusion protein can be detected. Pro-Q Sapphire 365 dye is excited by UV light, and its blue fluorescence can be visualized using standard UV transilluminators; Pro-Q Sapphire 488 dye can also be excited by UV light, but it is optimally excited by ~488 nm light, and its green fluorescence can be visualized using laser scanners. Both dyes are compatible with subsequent staining using SYPRO Ruby protein gel stain, permitting oligohistidine fusion protein and total-protein detection on the same gel.
Biotinylation of Other Biomolecules
Histidine, Serine and Threonine Modification
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 or sulfosuccinimidyl ester of biotin-X (B1582, B6353). The reaction probably involves formation of an acyl histidine intermediate, followed by intramolecular transfer of the label (Figure 4.7). 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 biotin hydrazine derivatives (B1603, B2600, D20653), which are described below.
Labeling with Biotin Hydrazides and Biotin Hydroxylamine (ARP)
As described in Section 3.2, aldehydes generated by periodate oxidation of vicinal diols in glycoproteins (Figure 9.49), polysaccharides and RNA or of N-terminal serine and threonine residues in proteins can be biotinylated using biotin-XX hydrazide (B2600). Biocytin hydrazide (B1603) reacts similarly and may be preferred because of its higher water solubility. As with our other DSB-X biotin reagents (Section 7.6), DSB-X biotin hydrazide (desthiobiotin-X hydrazide, D20653) can be used to produce a DSB-X biotin–labeled molecule that exhibits easily reversible binding to avidin- or streptavidin-labeled reagents. Biotin hydrazides have been used to quantitate periodate-oxidized glycoproteins on electroblots, as well as to biotinylate:
- Antibodies
- C
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