Analysis of disulfide bond formation.
In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein is then chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS-PAGE with and without prior reduction. The difference in mobility observed between the gels with unreduced and reduced samples is due to disulfide bonds in the unreduced protein.
Braakman
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I
University of Amsterdam, Academic Medical Center, The Netherlands.
Hebert
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DN
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002457:Cell Extracts; D002478:Cells, Cultured; D004220:Disulfides / Q000032:analysis / Q000378:metabolism*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism; D047468:Immunoprecipitation; D008861:Microsomes / Q000378:metabolism; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D011817:Rabbits
D002457:Cell Extracts; D004220:Disulfides