I am working with yeast saccharomyces cerevisiae -BY4741- strain. I tagged the Atg8 with GFP in this background and whenever I am performing western blot I get a free GFP signal for my WT which should not be the case. It seems like it's starving; I'm collecting my cells at OD600=1 and I'm inoculating them with a really low concentration. I'm adding all needed amino acids to my media and I performed different bead beating times to see if that makes a change. none of the above worked! can anyone please help me to solve this problem?
Hi Bahar Khonsari , could you please share a picture of your western blot that would be useful to give better suggestions. ATG8:GFP is the marker of autophagy vesicles and is surrounded by lots of proteases so, it is very important to make sure that protein degradation is not happening while extracting the proteins. By increasing the concentration of a cocktail of protease inhibitors in your extraction buffer, it would be possible to control degradation of proteins if it is happening during the extraction process.
Have you considered adding a low dose of Bafilomycin A1, 1 hour before extraction as a positive control ? It should stabilize your protein for Western Blots. If the cleavage persists despite Bafilomycin addition, then the problem could be with the extraction buffer as Sayanta Bera suggested.
Try to lyse the cells with RIPA buffer during mechanical shearing. Here is a link to the composition for Yeast lysis RIPA buffer. A strong SDS lysis buffer should keep protease mediated degradation to a minimum.
http://cshprotocols.cshlp.org/content/2011/1/pdb.rec12382.full?text_only=true
Thanks to both of you. Here is a picture of my gel the bands belong to BY without and with rapamycin treatment respectively. I've tried it for another yeast strain and I didn't get the free GFP signal, can it be that there is something wrong with my BY strain?
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