Glycoproteins biotinylation

The methods used for in vivo incorporation of azido-monomers and performing a labeling reaction with live cells are relatively simple. The following protocol is based on the methods of Saxon and Bertozzi (2000), which uses acetylated azidoacetylmannosamine as the azido-monomer source and a biotin-PEG-phosphine compound to biotinylate cell surface glycoproteins at the specific azide-sialic acid incorporation sites (Figure 17.19). 

The biotinylated glycans on the cell surfaces subsequently may be probed with (strept)avidin reagents to detect the azido-sialic acid modifications. Alternatively, the cells may be lysed and the glycoproteins isolated using an immobilized (strept)avidin or monomeric avidin affinity resin. 

Hydrazide-containing PEG-biotinylation reagents provide reactivity with carbonyl groups (e.g., aldehydes) to label carbohydrates or glycoproteins via hydrazone bond formation (Figures 18.19 and 18.20). The hydrazide group also may be coupled with carboxylate-containing... 

The following protocol describes a method for the periodate oxidation of a glycoprotein followed by biotinylation of the resultant aldehydes using hydrazide-PEG4-biotin. Chapter 1, Section 4.6 describes an alternative protocol for the modification of glycans at their reducing ends with hydrazide compounds. 

Figure 23.10 Glycoproteins may be oxidized with sodium periodate to generate aldehyde residues. These may be specifically labeled using a hydrazide-streptavidin derivative through hydrazone bond formation. Subsequent detection may be done using biotinylated enzymes.

Gretch, D.R., Suter, M., and Stinski, M.F. (1987) The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate herpesvirus hydrophobic proteins or glycoproteins. Anal. Biochem. 163, 270-277. 

After transfer, cut the membrane along the lane of prestained protein standards. Keep the piece of PVDF membrane with original, underivatized glycoproteins in a buffer for future elutions. Proceed with the detection of biotinylated glycoproteins. 

Block the piece of PVDF membrane containing biotinylated glycoproteins with 1 % (w/v) BSA in TBS for 1 h on a platform shaker... 

The low-molecular-weight vitamin biotin is easily conjugated to antibodies and enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule, and the strong affinity of the biotin for the glycoprotein avidin allows its use as complex-ing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) antibody can be used in the avidin-biotin methods. In the labeled avidin method the tracer is attached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylated enzyme such as peroxidase is allowed to bind after attachment of avidin to the biotin-labeled antibody. 

Figure 18 Glycoprotein-enrichment strategies for proteomics. (a) Lectin affinity chromatography (b) chemoenzymatic modification of 0-GlcNAc-modified proteins with biotinylated probes for proteomic analysis after avidin chromatography.

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