We need to analyze budding yeast cell extracts by Western blotting, and are looking for a good antibody against a "housekeeping" protein, serving as loading control (actin, tubulin, Gdh, etc.). Ideally, no vacuole protein.
Can anybody recommend commercially labeled antibodies that work?
There are excellent monoclonal antibodies that were originally raised against yeast alpha tubulin. These are from rat hybridomas and they are designated as clones YOL1/34 and YL1/2. They are commercially available from abcam (http://www.abcam.com/tubulin-yol134-antibody-microtubule-marker-ab6161.html
and
http://www.abcam.com/tubulin-yl12-antibody-loading-control-ab6160.html) but you can also find them by Serotec. I hope this helps
I have used the YOL 1/34 from Abcam not and verified that it does recognize yeast Tub1 (alpha tubulin). YL1/2 recognizes tyrosinated alpha tubulin but the levels of tyrosination can change in the cell.
Interestingly, the YOL1/34 and YL1/2 from Millipore do not react with S. cerevisiae tubulins, instead both recognizing a ~110 kDa band which I initially thought was a tubulin dimer. Although Millipore's antibodies seem to recognize something of a similar size as tubulin in mammalian cells, they do not recognize GFP-Tub1. Their tech support department refused to troubleshoot this problem on their end.
Monoclonal anti-beta actin clone C4 will recognize yeast Act1, another plausible option.
In my personal experience, what works best as a stable loading control is anti-PSTAIR or anti-PSTAIRE, which recognizes Cdc28 (Cdk1) and its paralog, Pho85. I buy ab10345 from Abcam for this purpose.
Mark is right. Both alpha tubulin clones are widely available from different companies. Although currently we are buying from Abcam, Serotec has proven a very reliable source of YOL1/34 since I started working with this antibody some 17 years ago. The C4 anti actin that Mark mentioned is also another excellent choice. I have not heard of this clone for quite some time now and it is good to know it is still marketed. Mark: do you have any company info on C4?
Hi George,
I ordered my C4 through MP Biologicals. It seems that Abcam and Millipore both carry it now as well. You are right, almost no one was selling it just 2-3 years ago.
Hi Mark, kudos for info. Actually the discussion on this actin antibody came around today and I was very glad to show the links you provided. Many thanks
Hi Maria.
In the lab we use the anti-3-phosphoglycerate kinase (PGK) mAb 22C5 from Molecular Probes, but it seems to have been discontinuated and now they have the clone 22C5D8. In our hands the 22C5 clone works more than fine at 1:10,000 dilution with a secondary antibody at 1:30,000 dilution, in TCA protein extracts.
The link for the new clone is
https://www.lifetechnologies.com/search/global/searchAction.action?query=anti+pgk&resultPage=1&resultsPerPage=15&autocomplete=
Hope it helps.
Hi Maria,
I don't know whether you need this control for normalization, but the use of such "housekeeping" proteins is probably not the best solution, as for qPCR.
You will probably find interesting alternatives following this link :
http://www.bio-rad.com/fr-fr/life-science-research/news/independent-study-reports-v3-western-workflow-with-stain-free-technology-yields-superior-western-blotting-results
And that article (specially in the "loading controls" paragraph)) :
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840294/
Good luck
Jean Luc
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