Hello everyone.
I am trying to detect phospho Akt in cell lysates of MZ-PC-1 and TT cancer cell lines, but I can't see anything at all. They should all have phosporilated Akt by default, as another laboratory that worked with them always had no problem visualizing it. I get a very nice band on commercial positive control cell lysate, but not in my samples.
I pellet my cells and lyse them in RIPA buffer with Pepstatin, MgCl2, PMSF, Roche Complete Mini cocktail as protease inhibitors, sodium orthovanadate and sodium fluoride as phosphatase inhibitors.
I then resuspend about 20-25 ug in NuPage LDS sample buffer with sample reducing buffer, denature at 95°C for 3 minutes, and perform the western blot.
I saturate the nitrocellulose membrane 1 hour with 5% milk TBST, then incubate overnight with Cell Signaling Ab #4060 in TBST 5% BSA, then an anti-rabbit HRP antibody 1:2000 for 1 hour.
I get a very nice positive control bad, but nothing in my samples. I've no idea what I am doing wrong. Maybe the inhibitors are not right?
Hi E. Galletta
Milk contains the phosphoprotein, caesin, which causes high background and non-specific binding when staining for phosphorylated proteins. This makes visualisation of phosphorylated proteins difficult. Use 5% BSA in TBST to block and 3% BSA with your antibodies staining solution instead.
You could also try an alternative source of lysis. CST do a complete cell lysis buffer which only needs the addition of PMSF - I would recommend repeating by their protocol:
https://www.cellsignal.co.uk/products/buffers-dyes/cell-lysis-buffer-10x/9803?_=1616677121707&Ntt=9803&tahead=true
https://www.cellsignal.co.uk/products/buffers-dyes/pmsf/8553
Finally, how many cells are you using and can you see your loading control? If your protein concentration is too low, you will not be able to visualise anything!
Best of luck!
Did you try to overexpose the film several hours or overnight? It's possible that Akt in those cell lines is weakly phosphorylated but not high compared to your control, but might still be positive. Especially if the secondary antibody is in a borderline concentration of being low.
I usually like to use Alkaline phosphatase-conjugated secondary antibodies because the signal is proportional to the time (at least until it gets saturated). HRP usually only last a few hours.
In the mean time, I'd repeat the experiment, follow Jack Hopkins ' advice , and add another set of cancer cells but treated with a growth factor that can activate Akt (say, insulin, FGF2, others reported in the literature) to make sure that you detect it next time in your cancer cells.
I agree with what Jack Hopkins said. Use 5% BSA in TBST rather than milk for blocking. Additionally, what concentration of total protein are you loading in your test sample? If the protein in your test is very less as compared to your control, it is highly possible that the weak band may not be getting detected despite being there. You can load higher or increasing concentrations of your sample or try some other cell line expressing p AKT rather than the commercial positive control cell lysate.
Exactly, replace milk with 5% BSA. And, load 30-40ug of total crude lysates, if possible.
I would also propose to modulate your method. Maybe BSA-based blocking soluion would be useful. Moreover, the specificity and efficacy of your antibody should be tested/evaluated.
Thank you for all your kind answers. I load 30 ug of proteins, and I load the same amount of positive control from CS. I can see a great band for the control, but nothing at all for my samples, that should be positive, according to the literature. Saturating with BSA did not change the result.
I can't expose my membrane overnight, I do not have the necessary equipment, nor other secondary antibodies.
I tried to change phosphatase inhibitors and lysis buffer, but nothing helped. Can the blotting conditions influence the outcome of my blot? If so, how?
I am not an expert in the field but just for curiosity, could it be an expression problem?
and,
Is it reasonable to catch and release the phosphoprotein by IMAC resins for both enrichment and clean up the protein from lysate followed by conducting a comparison assay with your positive control at PAGE platform?
In the case of a non-expression problem, you may exclude western for now and confirm the protein availability by performing both electrophoretic and chromatographic approaches in tandem.
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