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No band detected in WB - (Nov/03/2015 )

Hello all,


I'm new in this technique. I have tried to detect 180kDa protein in tissue sample. I already have protocol that can detect this protein and I have tried it many times. It's work. Now,my problem was there are nothing detected in membrane even if I used the same sample that used to detect in previous time. I have tried it for 4 times with same protocol and there're still nothing happen. 


Any ideas would be really appreciated.





are any of your solutions new since the procedure worked?


same lot of antibodies? stored correctly at all times?


it's not entirely clear, were the working detections also by western blot?


You can debug this (and all westerns) by working backwards. Spot the secondary in dilutions on a membrane, and test that your imaging reagents work well.  Now, spot your primary in dilutions, detect with the secondary, and test. Now, spot your samples in dilutions and detect with the primary and secondary. Only when this is working do you bother running a gel and transferring it to a membrane. It should be easy to determine what is not working with this technique.


Some proteins will degrade with freeze-thaws. Can you see your loading protein band? Have you confirmed that your transfer is working? 


High-molecular-weight proteins (>110 kDa) are notoriously tough to transfer.  What are your transfer conditions (semi-dry vs tank, voltage, time, temperature)?  What are the contents of your transfer buffer?  You may have to decrease the concentration of methanol and increase the transfer time to overnight.  You might even have to add 0.01% SDS to your transfer buffer (and leave out methanol entirely) if it is really stubborn.  If you are using semi-dry transfer, that method does not for proteins above 80 kDa.


And remember to stain your blot with Ponceau after the transfer to make sure it was efficient at your target molecular weight.