3 replies to this topic

[yoshimi79]

Does anyone have any tricks for Western blotting for Phospho-proteins? I'm looking at several different phosphorylated proteins in tissue samples and seeing a lot of variability.
Sample prep, Buffers, Transfer, freeze/thaw, boiling etc etc
I use protease phosphatase inhibitors in the homenization/lysis buffer, TBST and water to wash, and milk to block. Background is not an issue. Just highly variable signal

[Inmost sun]

yoshimi79, on Apr 30 2010, 04:17 PM, said:

Does anyone have any tricks for Western blotting for Phospho-proteins? I'm looking at several different phosphorylated proteins in tissue samples and seeing a lot of variability.
Sample prep, Buffers, Transfer, freeze/thaw, boiling etc etc
I use protease phosphatase inhibitors in the homenization/lysis buffer, TBST and water to wash, and milk to block. Background is not an issue. Just highly variable signal

what do you mean by variability? does the same sample of cell preparation give diffrent results? phosphosignal detection is more sensitive than normal protein staining by antibodies; one has to be careful with loading and amount of protein per lane

[yoshimi79]

The same sample can give variable results between runs. Nothing extreme, but the error seems to be larger with the phospho proteins. I don't always normalize the sample concentrations before loading...

[mdfenko]

i know you said that background is not a problem but milk contains a lot of phosphoprotein (casein) and may be having an effect on your western.

are you preparing your antibody solutions with block? the antibodies may be attenuated.

try using bsa to block.