Hi everyone,
I have problems with western blotting. Some times I will get clear bands and other times I will get nothing even though I ran the same sample and used the same blocking, and antibodies reagents and washing solutions. It was said to me that the problem could be in my cell lysing method ( although I keep my samples in sample buffer in -20 degrees) so I lysed directly in sample buffer to get as much proteins, sonicate and then when I ran a get I got this with tubulin. Any suggestions?
Thank you.
What about your time of blocking? Was it done in BSA or in milk? Did you use Protease inhibitor (PI) in your lysis buffer? How about boiling or heating the samples before you load them on the gel?
Also check the conditions of your buffers? What were the running conditions (Voltage etc) of the gel? Then we can reach to some conclusion
Dear Vikrant
I boil samples at 90 degrees for 10 min, spin for 2 min before loading in gel. blocking with milk in TBS. in this sample I lysed with sample buffer directly I did not add any PI but If I lysed with Lysis buffer I add PI. I used TGS running buffer at 70 V for 30 min then 180 V until the blue dye reach the bottom of the gel.
it looks like your protein is degrating somehow? Are you using PI? If I had to guess I also think there is a problem with your running glycine buffer... it doesnt even look so straight? I would make the fresh too (Not from the stock you have..) You can see that your protein is running weird.. ALSO the glas you use to run the western gel in clean it out well and make a fresh gel before running the samples.. ( I am assuming that you ran the exact same expt. again in the western blot so that shouldtn be the problem. I would focus on fixing the running conditions.. (ALSO dont run it on too high a Voltage)
Try to run initially at 50V until the protein enters resolving gel. In resolving gel run at 150V maximum. Do blocking with milk made in 1X TBST instead of TBS or IX PBST. for how much time have you done the blocking?
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