Hi all, I used water as my negative control in my immunoblot experiment, but i can see a band in this lane, could any one tell me why.
Whenever you see bands for your negative control, it generally means there was some form of contamination. However, it does depend on the size of the bands. If the bands for the negative control show products much smaller than the samples or positive control, it could possibly be primer dimers.
Hi Aries Huang
The is a possibility of non-specific antibody binding.
You could try the following.
1. Dilute the antibodies in (1X) TBS containing 0.1-0.5% Tween-20 in 3% BSA.
2. Increase the number of washes.
3. Use clean containers and freshly prepared buffers.
4. If you have enough lanes in the gel, you may leave one blank lane adjacent to the negative control lane.
5. Try to reduce the concentration of the secondary antibody.
6. Run a control with secondary antibody alone (omit primary antibody). If band develops, it indicates that you must use an alternative secondary antibody.
Best.
Hi Demos, thank you very much for your reply. I want to ask another question, this was a western blot experiment, why I can obtained a primer dimers band? As my understanding about primer dimer is related to qRT-PCR. Thank you very much!@Demos Kynigopoulos's
Malcolm Nobre Malcolm Nobre
Hi Malcolm, thank you very much for the suggestions. I'll try your suggestions and see what I can obtain later.
Thanks again!
Hi Aries Huang,
normally you don't see primer dimers in a western blot but its not impossible. If your sample before loading the column may be too concentrated this may lead to a 'forced' dimer that may not be natural. You can find more info in this previous thread https://www.researchgate.net/post/Dimer_at_sds-page
Best
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