Biotin conjugates

Reaction with avidin-labeled alkaline phosphatase (AP)-biotin conjugate... 

Although a number of cell lines were shown to be sensitive to inhibition by PatA (Low et al., 2005), we selected RKO cells (IC50 of 0.4 nM in cell proliferation assay) to prepare lysates for the isolation and identification of target protein(s). We often select RKO cells for target identification of small molecules using biotin-conjugates, because they appear to be particularly suitable for target protein isolation. 

Stain the gel with silver using standard procedures. Careful examination of the gel should reveal bands that appear in sample 2 and not sample 1, which can be assumed to be captured specifically by the biotin-conjugate. Furthermore, if these same bands are not present, or are lower in intensity in sample 3, they can be assumed to be specifically captured in sample 2, but lost in sample 3 due to occupation of the binding-site by the free compound during the pre-incubation step. 

Wilbur, D., Scott, C., Ming-Kuan, P., Pradip, M., and Hamlin, D.K. (2000) Biotin reagents for antibody pretargeting. 4. Selection of biotin conjugates for in vivo application based on their dissociation rate. Bioconj. Chem. 11, 569-583. 

The on-bead assay was conducted according to Scheme 3.19, which shows the chain of events, which leads to a colorimetric response, when an oligosaccharide binds effectively to the B. purpurea lectin. The lectin was covalently linked to biotin, a small molecule with an extremely high affinity for streptavidin. The bead-lectin-biotin conjugates were then exposed to streptavidin, linked to the enzyme alkaline phosphatase. Alkaline phosphatase hydrolyses phosphate esters [e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 110]. When the 5-bromo-4-chloro-3-hydroxyindole (111) is released, in the presence of nitro blue tetrazolium (NBT), it forms a dark purple, insoluble dye, thus staining beads where there was a favorable binding interaction. 

Streptavidin (Boehringer Mannheim, Mannheim, Germany) blocking buffer (1 pg/mL). Biotin-conjugated alkaline phosphatase (Boehringer Mannheim), in blocking buffer, 5 pg/mL. 

Add three drops of biotin-conjugated alkaline phosphatase (5 pg/mL) in blocking buffer to the slides and incubate at room temperature for 10 min. 

Qi K, Ma QG, Remsen EE, Clark CG, Wooley KL. Determination of the bioavailabiUty of biotin conjugated onto shell cross-Unked (SCK) nanoparticles. J Am Chem Soc... 

Not only biotinylated proteins, but also nucleic acids or peptides synthesized in the presence of biotin derivatives or carbohydrate-biotin conjugates, are separated by immobilized avidin and streptavidin, respectively, or are identified by avidin-enzyme or avidin-gold conjugates. 

Rohnnger, R. and Holden, D W (1985) Protein blotting detection of protein with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes Anal Biochem. 144, 118-127. 

Isotype-matched unconjugated, biotin-conjugated, or fluorochrome-labeled antibodies unreactive with mammalian cells are available as control reagents from many suppliers... 

Anti-mouse IgG (whole molecule)-biotin conjugate (Sigma-Aldrich, B-7264). 

Remove the supernatant and resuspend the cells in 10 pL PBS containing antimouse or anti-rabbit IgG biotin conjugate as required (1 10 dilution). Leave on ice for 10 min and wash the cells by addition of 180 pL PBS and centrifugation. 

As a first test of the QTL approach the viologen-biotin conjugate 4 was synthesized. Since the small molecule biotin is known to bind strongly and specifically... 

Biotin-conjugated rabbit anti-mouse 3. Avidin - biotin complex 4. Peroxidase conjugated biotin... 

Fluorescent immunoassay was also conducted. For instance, physisorption of goat anti-mouse IgG (biotin-conjugated) was first made to the PDMS surface for the immunoassay of IgG. Then neutravidin was introduced to bind to the biotin on the surface. Immobilization of biotinylated goat anti-human IgG on the neu-travidin-coated channel was first achieved by confining the reagent flow at a T-intersection on the PDMS chip. The antigen (Cy5-human IgG) was applied via a perpendicular channel. Fluorescence measurement allowed the... 

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