I am working on a protein which two homologs from this family is expressed in mouse, I have single knock out (KO) and I did RT-PCR and qPCR to show that the gene is not express anymore in my KO, however, I have problem to do western since my antibody is polyclonal and it could recognize the both proteins. I was wondering if anyone has suggestion for another technique instead of western?
Dear Marzieh,
For your experimental design, many things could be considered.
Although your antibody recognizes both proteins, if the molecular weight of your proteins are different enough, you might be able to make a distinction.
What about a 2D protein electrophoresis?
Best
Gilles
Can you tag any one homologue with GFP or something? If yes then you can perform the western using the antibody that you are using and antiGFP antibody. When you will use the other homologue antibody, there will be a band shift due to GFP tagging and when you use GFP antibody since it is tagged to only one of the homologue, only that will appear in the western. This will help you later in localisation studies as well..
All the best.
Dear Guilles,
Thanks for your answer. My proteins have almost same MW and it would be hard to make distinguish. Also, since one protein is deleted completely in animal at would be hard to interpret the results of the western.
Since there many protein with 14-17 KD are exist in the semen the polyclonal antibody can recognize the other proteins.
Best,
Marzieh
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