Hello everyone!
I was performing a WB for 3 months. First 2 months everything was ok and since 1 month I am not able to get good results. It looks like just random bands show up - at the correct MW, but still random - for example sometimes there is no band in control sample, where the protein is in this sample for sure... I use the same WB apparatus, the same transfer apparatus, the same transfer conditions (semi-dry transfer), the same buffers, the same primary (not the same exactly, but from the same stock) and secondary antibodies, the same dilutions... One day something just stopped working. Any suggestions? I am really confused.
Just wondering, how many times you freeze thaw your primary and secondary antibody? Try with new lot of antibody
When things go wrong...just throw everything and prepare fresh reagents. Results will come.
Just wondering, how many times you freeze thaw your primary and secondary antibody? Try with new lot of antibody
Yeah I am thinking about preparing everything from the beginning...
Primary antibodies were kept in 4 degree in 5% milk with 0,02% sodium azide. I use new portion since 1 month. Maybe I will try again with new solution. But we bought recently new antibody against another protein which I wanted to check and this new antibody didn't work - I dont think, that something is wrong with antibodies. Secondary antibodies are kept as a stock in 4 or -20 degree - depends on antibodies.
By random bands I mean that for example I have 4 samples in gel from one cell line and before in every case there was a band - now I can see, for example, bands only in sample 1 and 3 - and the situation is the same in case of every cell line, but with different samples.
I am repeating the experiments for better quality of results for standardization, so I have decreased total protein in cell lysate samples to 15 ug (in 10-wells geels), but I think that my problems started before it...and it would not explain this random bands...
you should use fresh buffers and reagents and also use freshly prepared antibodies.
you should increase the concentration of protein to 30ug and also run gel in duplicate
and perform cbb staining to find out whether your protein is degrade or not.
I had recently gone through the same. It should be your old Tris buffered saline (TBS) with tween 20 that is giving this problem. Throw all the solution and make fresh. Rarely do the secondary Ab conjugate like HRP is cleaved or its activity lost. This do happen. Your first option is to make all the solution again and if it still doesnt work, get a fresh aliquot of secondary and try it with the new solutions.
Maybe it`s not something stop working, but you may check your sample. In my experience, protein sample, even buffer added, could store at -20°c for 2 weeks, or it become clevage,which may lid to this phenomenon. I met the same thing before, after that, I only use protein sample prepared less than 2 weeks.
Thank you all for all the advice! I will try this week with new buffers and new samples! I really hope it will work!
I used primary antibodies in milk several times, but always stored at -20 ºC. Good luck!
I prepared totally new buffers, new antibodies and new samples... and it works again! So the conclusion is: if something worked and then stopped to work then you just should do this from the very beginning!
Thank you all!
most of the times, it;'s the primary antibodies that stops working.
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