Same samples but different results! - (Nov/09/2008 )
Hi,
I am currently trying to optimise western blots for my protein of interest.
Last week I ran a western and along with my samples I included a negative control which does not express my protein of interest. I decided to run two gels so I would have a back up, just in case anything happened to one. I diluted my samples in loading buffer and loaded half of it in one gel and the other half in the other gel. Both gels were then treated the exact same.
However when I developed, I got different results for the 2 different gels.
With both membranes I got bands I was expecting in my positive controls.
One membrane showed no band in my negative control lane, as I was expecting.
The other membrane however had quite a strong band, which appeared slightly lower than the band which I got with my positive control.
Is this likely to be unspecific binding? But why would this happen with one membrane and not the other?
I am just getting paranoid that the bands I have been detecting (which had taken ages & a lot of optimising to get) are not actually my protein of interest, although they appear to be approximately the right size.
Thanks for any advice.
Hey, don't panic, that's science! Even if you do everything the same, there can be something (sample swept over, impurities in the gel, etc) which changes the detection pattern, but do not worry to much about this, if it is just happening for the first time. Just go on with your routine and have an eye on this. If it is reoccuring, than you have to think about the problem.
Have you tried confirming that the bands you are getting are your target bands?
On western you can do this by peptide competition - get a synthetic peptide that matches the epitope of your antibody and pre-incubate your antibody with this before adding to a blot. In parallel run one as you normally would to make sure it isn't just a run issue. If the peptide blocks the antibody from binding to the bands you are seeing, then they are the right bands. If you can still see the bands, then they are non-specific.
Other methods could include RNAi to knockdown the RNA for your gene and hence the protein, only really works if you are doing cell culture though.
Immunoprecipitation - IP the protein from your lysis solution then run on a gel. Won't really tell you if your antibody is specific though.
No I havn't tried confirming that the bands I've been getting are my target bands. However my ultimate aim is to knock out my protein by RNAi, I just wanted to make sure I could identify my protein first before testing my suppressor molecules.
I think I will repeat this again and hopefully the previous result was a one off & I won't see it again.
Thank you both for your replies 