Dose anyone have any ideas about the high phosphor-protein level in my western blot basal (negative control)?
Our lab share the same protocol and almost the same regents. Some of the students haven't experienced this problem, but some have. The DMSO in basal is always under 0.1%. We have protease inhibitor in lysis buffer. We tried to always be gentle to the basal sample. Any idea why the basal is so easy to get phosphorylated for some certain protein (for example, its definetly high for pSmad2C but work very well for pAkt, I mean very low for pAkt)? Is phospho-protein samples even not stable in room temperature after denature (denature with sample buffer in 95 degree for 5 min.)?
Have you tried room temp PBS for basal? if you have this issue only for a certain antibody then may be you can try a higher concentration of agonist for stimulation?
Rizwana Afroz , yes, we can always try to increase the dose and treatment time of the stimuli to get response (the higher, the safer). I think in my experiment its not about the stimulation, cause I have done the experiment so many times, it works mostly. Like I said, high stimulation in basal still can happen for a unknown reason in some cases. And I personally prefer cold PBS. If someone can share their experience on handling sensitive proteins or any explanation to this phenomenon would be greatly helpful.
Ying Zhou I know Liam once used room temp PBS for reducing his basal.
Do you serum starve the cells for at least 12-16 hrs prior to experiment? Not sure about the details of your experiment.
You mentioned protein inhibitors but do you add phosphatase inhibitors? That would most likely help preserve your signal post lysis. Are the same samples picked up differently by different people? If so, could reflect different habits (reusing or not primaries etc...) and brainstorming might help figuring it out. Having high pX background isn't uncommon in my experience but is really a function of the Ab used. What detection method are you using (chemi, fluorescence etc...)? For fluorescence, sub-optimal processing of the image can significantly affect the data appearance.
Good luck!
Hi, Sébastien Soubeyrand , Thanks for helping us troubleshooting. We use chemiluminescence to detect the protein level. And we made lysis buffer with Na3VO4, NaF which are phosphatase inhibitors. It happens between people also sometimes happens if the experiments did by a single person. Not sure which step or handling is the critical one that leading to this.
Do you get inter person variability on the same sample? If that’s the case, human factors are likely. Do you have a pan Smad2C Ab? Do you see a band? That one is an obvious important control to have regardless of Phosphorylation and the corresponding signal should be seen consistently (not phosphorylation dependent). Just looked briefly at pSMAD Ab from Cell Signaling, a supplier that typically provides good quality Abs (not sure which supplier/which site you're looking at but one looks pretty good to me : Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb #3108), and they show readily feasible (drug based; common cell lines) positive controls. Given the challenges you’re facing, perhaps you should include a clear positive controls in your runs (HeLa +/- TGFbeta) routinely in your runs.
As NaF and Vanadate do not block all phosphatases it remains possible that other phosphatases are deP your protein. To mitigate this, you could consider lysis directly in SDS sample buffer, heating right away followed by bath sonication to eliminate viscosity.
Good luck!
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