Immunoblotting was performed using the following antibodies diluted in PBS-Tween buffer: α-myc 9E10 (Covance) at 1:10,000, α-Flag M2 (Sigma-Aldrich) at 1:3000, α-V5 (Thermo/Pierce) at 1:5000, α-GFP (Roche) at 1:1000 or α-GFP JL-8 (Clontech) at 1:5000, α-Pgk1 (Invitrogen) at 1:10,000, and α-S-tag (Thermo/Pierce) at 1:2000; α-Spc105 and α-Ndc80 antibodies were both used at 1:10,000 and were kind gifts from Arshad Desai (Akiyoshi et al. 2010 (link)). Immunoprecipitations were performed according to the kinetochore purification conditions using these antibodies conjugated to Protein G Dynabeads. Note that the images shown are cropped from original immunoblots for clarity.
Protocol detail
Immunoblotting and Immunoprecipitation Protocols
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Corresponding Organization :
Other organizations : Fred Hutch Cancer Center, University of Washington
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Variable analysis
independent variables
- Antibody dilutions used for immunoblotting
- Antibodies used for immunoprecipitation
- Protein expression levels
- Protein-protein interactions
- PBS-Tween buffer used for antibody dilutions
- Protein G Dynabeads used for immunoprecipitation
- Positive control: None specified
- Negative control: None specified
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