Problem: phospho signal quickly disappears

[amei]

Hi all,

I am having problems detecting phospho-signals, (ie. phospho PTEN,and phospho BAD)... they are diluted 1:1000 in 5% BSA/TBS-T.. and incubated overnight, 4 deg, gentle shaking. I got the bands but they disappear overtime, so exposing it longer doesn't help me... and sometimes.. two or three bands weren't even there... but I got strong signals with GAPDH for the loading control.
Can anyone give me any tips/suggestions?

Thank you in advance!

[admin]

Hi welcome
this is because you do not have enough phospho-protein to make a signal. You have two options:

1) re-add chemilluminescence and expose immediately - leaving the film in the case for 20-30 minutes without opening or moving it

2) re-do the western blot lysing in a SMALL volume of lysate, and adding phosphatase inhibitor - LOAD THE ENTIRE AMOUNT in the well - making sure you run and keep everything at 4 degrees celcius or on ice - even the gel running

I was able to get loads of signal using the above method even with difficult ones, also see this thread:
Western Blotting for Phosphorylated Proteins

best wishes

[amei]

Great! I am going to try those tips. Thank you!!!

[Cancer Biologist]

Hi there,

You got some good advice. In case it don't work, u should also try using stronger antibody concentrations, less blocking (i.e. dilute blocking buffer) and increase antibody exposure time. Westerns sometimes involve trial and error, just play around with the different variables/parameters in order to optimise the procedure and GET RESULTS. Hope this helps!

[Cancer Biologist]

Try using primary antibody dilutions of 1:500, see what happens. That's what I sometimes do with weak signals.