Western Blot question: cutting membrane into strips - (Jul/05/2005 )
I am trying to save up on my reagents, supplies, etc...and wanted to try cutting my membrane into strips and trying multiple dilutions on them for my next western. Just hoping someone has done this before and could shed some light on a few things i.e. if I should cut the membrane before or after my transfer, if I should cut the strips while the membrane is submerged in buffer...anything would be great help.
Thanks very much!
usually when i do this, i trnasfert all the gel on the same membrane and color it with ponceau red in order to view the lanes good. I cut at this time and de colour the mebrane. Then i do my blotting as normal.
Cut after transfer. Definitely use the Ponceau for visulaization. You can place the membrane on some saran wrap and cut on there. Since ponceau destains rapidly in water I wouldn't suggest leaving in the water while you are cutting.
Alternatively, if you have enough extract use a comb with a single well (or tape the combs together to form a single well) and load one large well with your sample. Then can cut membrane in strips without the worry of cutting down the middle of a lane.
If your lab has the money, and you plan on doing this alot, you can purchase a Miniblotter, Immunetic: http://www.immunetics.com carries them among others. I have used it quite a bit and saves time, protein, and antibody. But really, only worth it if you have alot of Abs to test.