Tips for Western Blot of Low Molecular Weight Protein - (Dec/02/2005 )
I am working on a project in which I am doing Western Blots to look at the expression of a low molecular weight protein (15 kDa) in various brain regions. I have been using a 0.2 uM nitrocellulose membrane for my transfers and I have been transferring for 1 hour at 100 Volts. I have been getting nice Ponceau staining, so my transer appears to be working. In addition, I have run a positive control with the purified form of my protein of interest and I have been getting nice results with that when I develop my film so my antibody seems to be working. However, I have not been able to detect my protein of interest in the brain. I was wondering if anyone had any tips regarding doing Westerns of low-molecular weight proteins. I've been using a 15% gel for SDS-PAGE and I am considering going to a 4-20% linear gradient gel. Will that help? Does anyone think I am having a sample preparation problem? I would appreciate anyones input. Thanks.
well, unless your antibody (raised against purified protein) does not work as well with native in situ protien (verrryyy long shot) then I would suggest that your gel - transfer -western setup is fine and you don't need to switch gel, transfer, or detection method or anything like that....
1. the only thing you might change is to switch to PVDF and fix your membrane after transferring, but that may not be a solution...this would help if you are washing off your protein during the western steps, but then you probably wouldn't see such nice results with your control
2. look hard at your extraction protocol. do you have a positive control for your extracts, that does not involve your POI? this is what I would check first. I bet there is a problem in your extraction method. I have not worked with brain tissue, so I can't offer any further suggestions except to say: carefully controlled pH, everything on ice, and protease inhibitors are your best friends....
One of the post-docs was working with a 8 kDa protein...using gradient gel, pvdf and the same transfer time as urs. She got nothing.
When I did a western for the same protein (it was kinda like a test for me i think, did not know that it had failed earlier)...i simply used a 15% gel, pvdf and a 30 min transfer at 100 V. Worked like a charm....
good luck...It also helps to make fresh transfer buffer(the same day as the western) and stick it in the 4C for it to be cold.
my colleague tell me that adding methanol in transfert buffer helps retaining the low mw proteins on membrane...
Your protein is not particularly low in terms of molecular weight, so using a 15% gel should be fine- there is probably no need to use a gradient gel for this. Are you sure you are using tissues that have the poi in abundance enough to be detected via Western?