Glyco-western blotting: biotinylated dermatan sulfate as a probe for the detection of dermatan sulfate binding proteins using western blotting.

摘要

Dermatan sulfate was biotinylated through the free amino residue of the core protein. The lysates of mouse and human lung culture cells were electrophoresed and blotted to a nitrocellulose membrane. The membrane was blocked successively with bovine serum albumin, avidin and biotin, and then treated with biotinylated dermatan sulfate followed by visualization using alkaline phosphatase conjugated avidin and its substrates. More than 30 bands were observed on the membrane when 1 microgram/ml of biotinylated dermatan sulfate was used. The binding was prevented by an excess of dermatan sulfate but not chondroitin sulfate A or heparan sulfate. Some of the bands resisted washing with high salt concentration buffer. 60 kDa heat shock protein was found to be a dermatan sulfate binding protein upon two dimensional electrophoresis.