I have run western blots and have got clean bands for AKT, phospho Akts and others but I am not able to get clear bands on repeating the western from the same samples around 3 weeks later.
My sample was C2C12 cell lysate.
Hello
agree with researchers
Make sure of the validity of the protein , and the protocol used of C2C12 cells
you can use this protocol :
cell seed in six-well plates at a density of 3 × 105 cells/well and incubated in GM for 24 h. Cells were rinsed with PBS once and incubated in DM. After 48 h, 100 μg/ml CHX and 1 μM MG132 or the same volume of DMSO (with which MG132 and AKTS stock solution was prepared) were added to DM, and cells were incubated for another 6 h before Western blotting. (B) Western blots of MG132- AKTStreated cells. Three independent samples were prepared for each treatment. Tbp was used as a loading control. (C) Phosphatase treatment of C2C12 nuclear protein; 5 μg of nuclear protein prepared from differentiating C2C12 cells without phosphatase inhibitor cocktail was treated with 10 units of calf intestinal alkaline phosphatase (CIP) in the absence or presence of phosphatase inhibitor cocktail (PIC) for 10 min at 37°C and analyzed by Western blotting using a 7.5% gel.
Good Luck
Keep your samples at -20C or -80C and try again. Hope your problem will be solved.
Well, the question is do you have trouble in reproducing the both Akt and p-Akt or is the problem with only p-Akt. If the former case, protease inhibitors and storage at -20C might help. Additionally, it is important to heat the samples in sample buffer prior to storage. This will eliminate any proteolytic degradation.
If case of latter problem, it is important to add phosphatase inhibitors as suggested above. Once again, heating the sample in sample buffer will prevent further dephospho rylation. On the day of your experiment, take an aliquot for protein assay and heat the rest of the sample in sample buffer prior to storage.
...and dont forget "using phosphatase inhibitors in the lysis buffer: combination of phosphatase inhibitors, NAF, Na orthovanadate, Na deoxycholate, Na pyrophosphate along with protease inhibitors (RIPA buffer with all inhibitors)....this is VERY IMPORTANT...!!!
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