I can successfully probe for 1 protein but NOTHING else after (actin, gapdh or tubulin). I combined the primary antibodies of actin and my protein of interest and this seems to skirt around the problem. Though, I'm curious to see if anyone else has had this issue and if they nailed the culprit(s). FYI, I use laemmli lysis buffer, sonicate the cells, 10% sds-gel (made with new reagents), Ponceau staining looks fine (image attached) on nitrocellulose membrane, block in 3% milk and probe with primary antibody in BSA, and we have new ECL detection reagents. I've tried increasing the ratio of primary to BSA and nothing works. What gives?
Western blot membranes which were allowed to dry during the first blotting, does not work for striping and reprobing. youl should be carefull and keep membranes in washing saline buffer before and after striping. Also you should add some bacteriostatic solution to avoid membane contaminations, 10-20 ul of thimerosal works well.
Also check he dilutions of your primary antibodies. Santa cruz antibodies in particular work usually well at high dilutions.
Actually the problem is with using nitrocellulose mambrane. You can use PVDF membrane instead.
The two most common membrane types used for western blots are nitrocellulose and polyvinylidene fluoride (PVDF). Nitrocellulose was one of the first membrane types used for western blotting. Its advantages are that it is easily wetted with an alchohol solution and it produces good immunoblotting results. PVDF membranes were developed in 1985 and exhibit improved protein retention under harsh conditions (i.e. in the presence of organic solvents or under acidic or basic conditions). The greater mechanical strength of PVDF membranes is an asset when handling the membrane compared to nitrocellulose. In addition to its chemical stability, PVDF offers advantages when stripping and reprobing in immunodetection applications.
Ref: http://www.pall.com/main/laboratory/literature-library-details.page?id=48923
how do you do your transfers and how do you store your membranes? Did you try PVDF?
Thanks for all the thoughtful suggestions, I will give them a try.
Anwar, we use a wet transfer and store membranes in PBST wash buffer. I have not tried PVDF because our lab only uses nitrocellulose.
I usually use two antibodies (one internal control HSP70 and other one specific antibody) but usually I use antibodies raised in different species. For example if I use control antibody from mouse I use specific antibody from other species like rabbit. Usually it work perfectly in nitrocellulose membrane.
- Badges
- Science method