Santa Cruz Biotechnology
Gα s Antibody
sc-823
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The Gα s subunit of the heterotrimeric G proteins stimulates adenylate cyclases to generate cAMP, an important second messenger molecule that is necessary for PKA activation. The goal of this experiment is to test if NIH3T3 cells express Gα s. If it is expressed, I would then go on to determine the localization of this protein. The reason for selecting this product from Santa Cruz is that it was referenced in few publications.
Western Blot
NIH3T3 cells_50 µg of whole cell extract
1:200 in blocking agent for 16 hr at 4 deg
5% milk powder in 1x TBS-T (0.1% Tween-20)
Donkey anti-Rabbit IgG HRP
NA
ECL pico for 5 min
This antibody detects a band at ~46 kDa (expected size) but also detects few other bands at 50 and 55 kDa. The signal is poor and one needs ECP pico or ECL femto. I had tried changing milk powder with BSA in blocking buffer but did not improve the result. I had also tried primary antibody incubation for 1 hr at room temperature but even that did not improve the results.
None
OK antibody
Poor signal with many background bands
Not the best in the market