Western Blotting - (Jul/31/2002 )
I'm a new bird in doing western blotting. I just want to ask:
1. besides cossmassie blue staining the SDS-gel, can I
check the transfer efficiency by staining the PVDF
membrane? Also, what sort staining solution
recommended for this purpose?
2. what is the different between using PBS based and
TBS based buffer in the blocking and washig step of
antibody labelling? And is there any differnece for
ommitting Na azide in the washing buffer?
Thank for helping me to slove these puzzles.
You can view your transferred proteins by reverseable staining of your membrane with Ponceau C or Amido Black. You can find protocols for this on the web. I haven't tried this myself (I'm a newbie too) but staining the membrane this way apparently doesn't affect detection.
I've just started doing Westerns too. One problem I am having is in making the gels - I often get a thin film of gel in the wells in the stacking gel. I've tried flushing this out and also scraping it out with a needle but with little success. Anyone else had similar problems?
I've used Ponceau stain in the past to stain the membrane with good results.
How many cells are people using per sample? I'm trying 2m (lymphoma cell line) but I've read that I should be using about ten times that amount. Problem with that is the culture volumes I usually use are small. I'm inducing apoptosis in these cultures so I have to subculture the day before.
As a pseudo-veteran of the immunoblot world, I can offer you a couple of insights I have learned along the way:
1. The best way to determine transfer efficiency to stain your PVDF membrane with Ponceau S. It is both sensitive and reversible. Amido black, India ink and colloidal gold staining can also be used but these stains are not reversible and MAY interfere with antibody binding.
2. Either TBS or PBS-based buffers can be for immunolabeling. Some antibodies work better in TBS while others work better in PBS. I typically use PBS plus 0.1% Tween-20 for immunoblot analysis.
3. If you are using an HRP (horseradish peroxidase)-based detection system, then you MUST omit azide from ALL of your buffers. Azide will inhibit HRP activity.
Hope these pointers help.
Thank alot for you guys suggestions and I'll try all these. I'll tell what's the results after tried. Thanks once again.