What kind of P-protein(s) are you detecting?  We routinely use 6-well plates for detecting any P-proteins such as Erk, Akt, Smads, beta-arrestin etc in WB.  The low level of detection may reflect your lysis buffer or Abs used. 

Some of those you list! What amounts of total protein do you load to detect phospho erk and AKT please? Any ideas on whether centrifuge concentration of samples will affect the proteins phosphorylation please? 

I don't bother to measure proteins, as I routinely re-probe total protein after each P-protein, and never get uneven issue.  The key is to seed your cells into 6-plate wells one well at a time after shaking (e.g., 2 ml of appropriately diluted cell suspension say 40,000 cells/ml).  Technically, centrifugation wouldn't affect your phospho-protein, but optimization of your WB system may dramatically increase your detection sensitivity.

That's great, thanks, I'm trying phospho protein detection for the first time, had no issues with blotting for total proteins. Idea behind using t75 flask for protein is to use same lysates for many different blots without the hassle of having to repeatedly grow/plate/harvest cells

Hi Craig,

I routinely use Millipore's Amicon series of centrifuge concentrators on phosphoprotein samples followed by immunoblotting with anti-pThr and anti-pY antibodies (also Pro-Q staining).  The concentration step is unlikely to affect phospho-status of the proteins in your sample (phosphorylation is quite stable) provided you have adequate inhibition of phosphatase activity in your lysis buffer.

Thanks Kyle, my main worry is that in the system I'd like to use some heat is generated as a by-product of the centrifuge, any ideas if this heat will effect the phosphorylation state (over and above heat in general not being great for protein structures...)

As long as you aren't hitting very high temperatures for long periods of time your samples should be fine.  I run my concentrators in a refrigerated centrifuge at 10 degrees Celsius. 

The lysates (0.3 ml) from 6-well plates can be used for many WB blots (more than 10, as each lane uses only 0.025 ml).  Another advantage of using smaller wells is to minimize any treatment agent(s), some of which are expensive. 

Thanks very much kyle and ke-Wei, your advice has been very useful!

Have you considered concentrating the sample by using antibodies to pull-down the protein of interest? Resolve the pellet by SDS-PAGE and then detect protein of interest by Western blot using phospho-Ser/Thr or phospho-Tyr antibodies.

That's an interesting suggestion, thanks Susan!

An added point to Susan's IP suggestion:  You can do one IP at a time and are tied up with the vast number of samples.  Also, the use of anti- P-Ser/Thr or P-Tyr Abs seem to be out dated, as mAbs against  site-specific phosphorylation of a particular protein are readily available and widely acceptable.  For example, there are two P-Akt mAbs, T308 and S473, each recognizing phosphates at respective site.  Practically, one has to use expensive high quality BSA to block PVDF membranes, but can use powdered milk (containing lots of P-Ser/Thr and P-Tyr proteins) in the mAb cases!  It saved us thousands of dollars on BSA alone, which helped us getting a range of mAbs against most P-molecules.