A UK-based reader writes
I'm a UK molecular biologist with DNA oriented experience who is about to try a western blot for the first time. Are you willing to share your protocol?
To be honest, it's pretty much the standard protocol. The antibody isn't fantastic (although that might be a function of my protein, of course) and I used to get a lot of background, but here's a quick guide to getting this particular antibody against this particular epitope in these particular samples to work (standard disclaimers, caveat emptor, this advice is worth exactly what you pay for it, etc. ad nauseam). I've emphasized the points that needed to be optimized:
- Prepare protein from cells (it was a nuclear protein, so I used this method up to and including step 6)
- Run gel (I used Invitrogen NuPAGE pre-cast Bis-Tris gels in
MOPSMES running buffer) - Soak gel in Towbin buffer (Tris/Glycine/20 % MeOH) for 10 - 15 minutes
- Blot onto PVDF at 200 mA for 2h at 4°C
- Block in 3 - 6% milk powder in PBS/0.02% Tween20 overnight at 4°C
- Wash membrane 3 – 6 x 5 minutes in PBS/0.1% Tween20 (PBS-T)
- Incubate in primary antibody for 3 – 4 hours at room temperature (you'll have to work out the optimal concentration yourself)
- Wash in PBS-T
- Incubate in secondary antibody diluted 1/20,000, no more than 30 minutes
- Wash 6 x 5 minutes in PBS-T
- Develop signal with Pierce Femto substrate (1.5 ml each reagent per membrane) for 1 minute. Pat dry. Leave 5 minutes for signal to burn off a bit
- Expose and develop film, whoop loudly.
The primary and secondary antibody diluent is PBS/0.05% BSA.
My friendly reader also said
I have to develop protocols for antibody purification- possibly involving formaldehyde crosslinking- can you give me any advice?
and I do have a protocol for that, here in fact, and am happy to answer questions on it.
