Hi all,
I am transferring the proteins (extracted from sperm in Laemmli buffer) to a 0.22µm nitrocellulose membrane at 100V for 60 min (wet-transfer). However, Coomassie blue gel staining after electro-transfer shows the presence of protein bands. Is it normal to not have all the protein transferred to the membrane?
Although, I am targetting for lower molecular weight proteins which are mostly transferred, however, large molecular weight protein doesn't transfer well.
Hello Saurabh Tiwari
You may use the method given in the articles below for the transfer of high and low molecular weight proteins to nitrocellulose membrane.
References:
Article Western Blotting of High and Low Molecular Weight Proteins Using Heat
Article A two-step procedure for efficient electrotransfer of both h...
Best.
It's not shocking, large proteins will take a little more time to transfer since they have to pass the gel matrix and it takes more time for them. I know that we transfer a bit longer (90 min) at 200 mA, but yes, it can definitely happen.
