What is the purpose of biotinylation?

The biotin–avidin interaction is commonly exploited to detect and/or purify proteins because of the high specificity that these two molecules have for each other. Biotinylation is the process of attaching biotin to proteins and other macromolecules.

How are biotinylated antibodies made?

Biotinylated antibodies are used in two methods: Avidin-biotin complex (ABC) method: large avidin-biotin complexes linked through reporter enzymes are incubated with biotinylated antibodies. The signal is amplified due to the high enzyme-to-antibody ratio.

What is Surface biotinylation?

Cell-surface biotinylation is one of the most commonly used methods for isolation of plasma membrane proteins, in which the extracellular domains of integral and plasma membrane-associated proteins are covalently labeled with a reactive biotin ester.

What is histone biotinylation?

Biotinylation of histones suggests that the vitamin biotin might play some role in transcription, replication, or repair of DNA in analogy to the effects of acetylation, methylation, phosphorylation, ubiquitination, and ADP-ribosylation of histones.

What is biotin in biotinylation?

In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to disturb the natural function of the molecule due to the small size of biotin (MW = 244.31 g/mol).

Why is biotin used for labeling?

It is widely used to label a variety of biological molecules and samples. Red cells are labeled with the spacered biotin, and the labeled cells are detected in small blood samples with flow cytometry. Improved labeling efficiency and binding affinity allow the easy detection of positive red cells.

What is a biotinylated antibody?

Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents.

How does cell surface biotinylation work?

At this temperature, biotinylated surface proteins are endocytosed. Following incubation, the cells are cooled to 4°C, and a hydrophilic reducing agent like L-glutathione is added. This reacts with disulfide bonds and releases the biotin groups from labeled, unendocytosed proteins.

How do you Biotinylate a protein?

Besides whole proteins, biotinylated peptides can be synthesized by introducing a cysteine (Cys) residue during synthesis at the terminus of the amino acid chain to get a site specific and oriented biotinylation. Nucleotides can also be biotinylated by incorporation of thiolated nucleotides.

Why is biotinylated secondary antibody used in ELISA?

Secondary antibodies labeled with multiple biotin molecules, when combined with a streptavidin- or avidin-based conjugates, allow for signal amplification of lowly expressed proteins.