Biotin maleimide

If a free amino group forms a portion of the protein that is essential for activity (e.g., the antigen-combining site for antibody), biotinylation with the succinimide ester will lower or destroy the activity of the protein, and other methods of labeling should be tried. Biotin hydrazide has been used to modify the carbohydrate moieties of antibodies (10,11). Other alternatives are the thiol-reactive biotin maleimide (12) or biotin iodoacetamide (13). 

Prepare the antibody for reduction. For labeling IgG antibodies with thiol-reactive reagents, the antibody should be at 5-10 mg of pro-tein/mL in 20 mM sodium or potassium phosphate buffer, pH 7.5-8.0 containing 150 mill NaCl or 10 mM phosphate-buffered saline ethylene diamine tetraacetic acid (PBS-EDTA). When biotin maleimide or biocytin maleimide derivatives are used, the buffer used for the reduction and subsequent steps should be at pH 6.5-7.2. 

Biotin-maleimide (available from Sigma) has been conjugated to deprotected Cys-tagged proteins using a combination of methods described in Subheading... 

For the dienophile some structural requirements are necessary for acceptance by the ribozyme as a substrate (Figure 5.3.7). It must be a five-membered ring and substituents on the reactive double bond are not permitted. The biotinyl residue is not necessary for recognition of maleimide by the 49nt RNA, because the initially used biotin maleimide can be truncated to a maleimide and an alkyl chain, both of... 

The K138C mutants (-1.5 mg in 0.5 mL of PBS) were treated with 50 mM DTT (removed by gel filtration) and then biotinylated using biotin-maleimide (Sigma) (2 1 molar ratio). The biotinylated product was purified by gel filtration on Superdex 75. Evidence of biotinylation was routinely obtained by western blots probed with an avidin-HRP conjugate (Pierce) and by binding of the proteins to streptavidin-coated BIAcore chips (Pharmacia SA5). 

Diels-Alderase ribozymes (DAR), isolated from a combinatorial RNA library, cause a (2 X 10 )-fold acceleration of the Diels-Alder cycloaddition of anthracene covalently tethered to ribozyme and a biotinylated maleimide in aqueous-buffered medium (Scheme 5.15). Jaschke recently reported the action of Diels-Alderase ribozymes as true catalysts, in the sense that they catalyze the cycloaddition of anthracene that is not covalently tethered to RNA and biotin maleimide in aqueous-buffered medium. 

IR point irradiation of the FMP-functionalized 3D structures in the presence of a biotin maleimide solution ... 

Figure 11.8 Biotin-BMCC provides sulfhydryl reactivity through its terminal maleimide group. The reaction creates a stable thioether linkage.

The reagent is similar to another maleimide-containing biotinylation reagent, 3-(N-maleimi-dopropionyl) biocytin, a compound used to detect sulfhydryl-containing molecules on nitrocellulose blots after SDS-electrophoresis separation (Bayer et al., 1987). Biotin-BMCC should be useful in similar detection procedures. 

Diene-modified oligo Maleimide-PEG2-Biotin... 

Figure 17.2 Maleimide groups provide good dienophiles for a Diels-Alder reaction. Biotin-PEG2-maleimide can react with an oligo-diene molecule to form a covalent cycloaddition product, which adds the biotin tag to the oligo.

Figure 17.3 Maleimide-modified glass slides (1) can be derivatized using two chemoselective ligation reactions to create biotin modifications. In the first step, alkyne-PEG4-cyclopentadiene linkers (2) are added to the maleimide groups using a Diels-Alder reaction. In the second reaction, an azido-PEG4-biotin compound (3) is reacted with the terminal alkyne on the slide using click chemistry to result in another cycloaddition product, a triazole ring.

The three maleimide-PEG -biotin compounds illustrated in this section provide short, medium, and very long chain spacer options, with the longer chains resulting in the greatest degree of hydrophilicity of modified molecules. The following protocol is adapted from general maleimide-based biotinylation methods, as discussed in Chapter 11, Section 2. 

Figure 18.17 Maleimide-PEG -biotin compounds of three different discrete PEG sizes are available, including a PEGn chain that provides a molecular length of over 60 A.

Prepare a stock solution of the maleimide-PEG -biotin compound in DMAC, DMSO, or DMF (pure and dry solvents only) at a concentration of 10-20 mM. 

Figure 18.18 Maleimide-PEG -biotin compounds react with thiol-containing molecules to form thioether linkages.

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mer-captoethylamine and creates a thiol group for conjugation (Figure 27.6). DNA probes labeled in this manner have been successfully coupled with SPDP-activated alkaline phosphatase (Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP) (Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5). 

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