My target is phospho-TCS2 in AKT/mTOR signaling pathway. The western is done on total cell lysate prepared from human cell lines and I am using phospho-TCS2 antibody from cell signaling.
Can you upload an image of the western's you've gotten so far?
A couple suggestions:
1. Block in milk, rinse twice, and probe overnight at 4C in:
a) BSA
b) non protein blocking agent (like Superblock from Thermo)
Some phospho antibodies (all pS6K1 T389 antibodies from Cell Signal for example) give a much better signal to noise ratio when blocked in milk and probed in BSA or nonprotein block.
2. TSC2 is relatively big, try reducing the methanol in your transfer to 5%
Those should help but without seeing the blot it's hard to know. Best of luck.
cb
We use in Odyssey Blocking Buffer diluted 1:1 with PBS/0.1% Tween block for 1 hour and then incubate the antibody in that same buffer. Make sure you have added plenty of phospho inhibitors.
Dear Chris and Amy, thank you for your response. Actually, I did not have any problem with lower molecular weight in mTOR pathway. As Samantha has preposed in her comment, the key point was using wet transfer instead of iBlot system to transfer the proteins from the gel to the membrane. Just by this change, I was able to get the result. Thank you all.
Hi Chris,
Did you probe with primary and secondary antibodies diluting in superblock together with blocking in milk? May I know the outcome of the result? Did they enhance the sensitivity of the bands of interest, but not showing other non - specific bands on your membrane?
Thanks.
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