I am doing some Western Blots and I want to trace bands of the phosphorylated p70S6K in mice brain tissue. I have repeated all my experiments more than 4 times and I still get no or very low density bands. My experiments concern the mTOR signaling pathway and although p-mTOR works perfectly, I have this constant problem with the kinase. This kinase is supposed to be a downstream protein in this pathway but although mTOR shows different bands, the kinase doesn't follow the same patterns. The abs I'm using are from Cell Signaling with a 1:1000 dilution in 5%BSA. The only difference is that the ab for p-mTOR is XP but the one for the S6K is not. Do you perhaps have some suggestions on what I should do or if there are any better abs in the market? Or is there any step in the protocol that I should be more aware of (like incubation time, dilution, etc.)?
Thank you all in advance!
Load 40-60ug protein/well. 10% gel. Use nitrocellulose membrane for transfer, use 10% milk for blocking and for reconstituting the antibody. In parellel, use total p70S6K to make sure that the sample you use has p70S6K expression. Good luck.
Also, make sure the lysis buffer has phosphatase inhibitor in it. Additionally, I've found that boiling for a shorter time improves detection of some phosphorylated proteins, including Thr 389 on p70S6k. I boil for 1 minute, and also use the cell signaling antibody, and it works well in heart samples. i use hybond-p membranes from amersham and the ECl kit from amersham.
I have different experience about blocking. I always use 1% BSA in TBS-T to block for phospho-proteins after struggle with 5-10% skim milk blocking for years. In endothelial cells I was able to detect strong P-P70S6K with 30 ug/well cell lysates. Abs from Cell Signaling were used in my study.
By the way I forgot to mention that I use precast BioRad gels which I think are 7,5%. Do you think that a homemade gel would be better in this case? Although I have no problem with all other proteins. I was also thinking to do an IP in case my samples are of low concentration of this specific phosphorylated protein. Maybe more ug of the protein could also work..
Do you have a positive control? It would be beneficial to have a sample subjected to an acute insulin treatment (ICV or IP) to determine if it is the antibody, the model, or something else impacting your results. I have had success using the Cell Signaling p70 Ab's.
Hello ,
I'm also working with HT-29 cells on mTORC1 pathway . My primary focus is the p-P70S6K protein which is really difficult to work with. I have tried loading 150-170ug of protein (it's always difficult to produce so much protein and electrophoresis is difficult with large amount of protein). I'm using Lycor nitrocellulose membrane for blotting and milk (5%) is used for blocking the membrane. I had been using HRP-linked secondary antibodies for detection but got very faint signal so I recently shifted to lycor IR antibodies but the success is very variable and limited. I'm using primary antibody from cell signalling (Thr 389) which is a rabbit monoclonal antibody.
Kindly suggest something.
To Aimilia, I do not think precast gel is the problem since that was what I used before. I was able to see the signal at 30 ug/well in pulmonary artery endothelial cells in fetal sheep. Surely you can try IP if the signal is really low. I have some experience that one batch antibody work whereas the other batch from the same vendor did not. Another question is how abundant is the p70S6K in your cell which I do not know.
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