S100 cytosolic extracts were prepared from HEK293 vector and SIRT2 knockdown cell lines. Equal amounts of protein were subjected to immunoprecipitation with anti-acetyl lysine antibody. The proteins were eluted with glycine buffer (pH 2.7). The proteins were then subjected to in vitro labeling with Cy-3 and Cy-5 N-hydroxysuccinimidyl ester. Cy-2 was used as an internal standard. The samples were subjected to isoelectric focusing and then separated in a second dimension by SDS PAGE. The gels were fixed, stained and protein spots were analyzed using GE Healthcare DeCyder software. The protein spots of interest were subjected to automated in-gel tryptic digestion and MALDI/MS/MS spectra were performed with 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems) (24 (link),25 (link)).
Protocol detail
Quantitative Proteomic Analysis of SIRT2 Knockdown
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Anti antibody
Buffer
Cell lines
Cytosolic
Digestion
Ester
Glycine
Immunoprecipitation
Lysine
Maldi
Protein
S100
Sds page
Sirt2
Spots
Tryptic
Vector
Corresponding Organization : The University of Texas MD Anderson Cancer Center
Other organizations : FuelCell Energy (United States), Georgia Regents Medical Center, Augusta University, University of Oklahoma Health Sciences Center, Baylor College of Medicine, Johns Hopkins Medicine, Johns Hopkins University, Cornell University
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Variable analysis
independent variables
- Knockdown of SIRT2 gene in HEK293 cell line
- Acetylation levels of proteins detected by immunoprecipitation with anti-acetyl lysine antibody
- Protein expression levels detected by 2D gel electrophoresis and mass spectrometry analysis
- Equal amounts of protein used for immunoprecipitation
- Cy-2 used as an internal standard for 2D gel electrophoresis
- Positive control: HEK293 vector cell line
- Negative control: Not explicitly mentioned
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