High-throughput protein extraction and immunoblotting analysis in Saccharomyces cerevisiae.
A variety of Saccharomyces cerevisiae strain libraries allow for systematic analysis of strains bearing gene deletions, repressible genes, overexpressed genes, or modified genes on a genome-wide scale. Here we introduce a method for culturing yeast strains in 96-well format to achieve log-phase growth and a high-throughput technique for generating whole-cell protein extracts from these cultures using sodium dodecyl sulfate and heat lysis. We subsequently describe a procedure to analyze these whole-cell extracts by immunoblotting for alkaline phosphatase and carboxypeptidase yscS to identify strains with defects in protein transport pathways or protein glycosylation. These methods should be readily adaptable to many different areas of interest.
Lorenz
Todd C
TC
Department of Biological Chemistry, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
Anand
Vikram C
VC
Payne
Gregory S
GS
D000469:Alkaline Phosphatase / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D002268:Carboxypeptidases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D005800:Genes, Fungal; D006031:Glycosylation; D015151:Immunoblotting / Q000379:methods*; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification*
D029701:Saccharomyces cerevisiae Proteins; D000469:Alkaline Phosphatase; D002268:Carboxypeptidases