Hi Shyam, what was the culture condition you used? I usually culture yeast cells in 3-5ml drop-out medium for o/n, collect them by centrifugation and directly boil cells with SDS sample buffer. After boiling, just load the supernatant into gel instead of whole cell lysates. Remember don't grow yeast in YPDA because yeast will throw your plasmids away since the growth environment is rich.  

Ya,I grew them overnight in 10 ml dropout media up to OD600=~1,and then used glass beads for recovery of protein from cells using boiling method without adding any protease inhibitors. I tried standard GAL based antibodies as well as specific antibodies but not getting any significant or repeatable success.  Are you also used Invitrogen proquest kit ?

Thank you very much for answer, I appreciate that.

Hi Shyam,

I proceed as described in: Kushnirov (2000) Yeast 2000; 16: 857±860.

"Rapid and reliable protein extraction from yeast"

That is what they do (it worked for me):

"For the novel extraction protocol, designated here as post-alkaline extraction, about 2.5 OD600 (which constitutes about 2.3 mg of wet weight) of yeast cells were harvested by centrifugation from liquid culture or scraped off the agar plate using a bacteriological loop. These cells were resuspended in 100 ul distilled water, added 100 ul 0.2 M NaOH, incubated for 5 min at room temperature, pelleted, resuspended in 50 ul SDS sample buffer, boiled for 3 min and pelleted again. About 6 ul supernatant was typically loaded per lane of SDS-PAGE gel. The sample buffer (0.06 M Tris±HCl, pH 6.8, 5% glycerol, 2% SDS, 4% bmercaptoethanol, 0.0025% bromophenol blue) was slightly modified from standard Laemmli."

For my experiments, I replaced mecapto by DTT as a reducing agent.

Hope it helps in case it hasn't worked yet.

Thank you Stella, I was able to get results in westerns  using a bead based protein extraction protocol. Suggested protocol looks much easier and I will definitely give it a shot.