Is there a problem? Our group use regular western blot and was able to obtain decent signals in our AMPK paper. We did not do anything special. 

I want to know what are the transfer times you used so the voltage settings .

We used wet transfer 400 mA for 60-75 minutes on ice (somebody uses 100V for 60 minutes but we do not). If you want to see phospho proteins then block with 1-5% albumin in TBS-T.

I mean mTor bei g of 290 kd did you transfer it at 400 ma at 60-75.inutes?  Also did you use 6 %gel a d may i have link to your paper

Yes, we used 6% gel. Our power pack actually can not go above 400 mA. Our model is sheep pulmonary artery endothelial cell and CellSignaling antibodies work well. The full article is available in ResearchGate but am happy to provide the link as below.

http://ajplung.physiology.org/content/ajplung/304/1/L29.full.pdf

My Westernblotting conditions for p-mTOR and t-mTOR are as follows and work well:

(1)7.5% SDS-PAGE gel

(2)Transfer voltage: 100V for 120min.

(3)Block: 5% milk for 1-2hr.

(4)Primary antibody incubation: 1:2000 antibody (CST) at 4℃ >12hr.

(5)Secondary antibody incubation: 1:5000 antibody at RT 1-2hr.

Hope to be helpful.

In the care of renal cell carcinoma(786-O) transfer condition?or nc membrane?

Hi Ru-Jeng Teng, have you ever lost your smaller size protein (10-20 kDa) when you transfer @ 400mA for more than 1.5 hours?

Also what is in your transfer buffer?

Thanks!