I just have a couple of questions. I am starting some western blots of my protein extracts and I want to know how many times i can re-use a mabrane and what is the best way to strip them.
I am using 0.45 um nitrocellulose mebrane and I have started work on each membrane by immunostaining for beta actin (~ 43 kb) to ensure equal protein concentrations and good sample separation.
I also have to stain for about 6 or 7 more protein markers of my cells which are all close enough in size to the beta actin marker (~ 35 - 60 kb)
So can anybody tell me how many times i could strip one of my better quality membrane after beta acting staining and what is the best way to do it?o
I have tried a technique using beta mercaptoetahnol and other ingredients and heating to 60 C for 30 mins and then washing and reblocking but have read that this degrades the membrane, I have also heard of another technique washing with NaOH only.
A colleague who did WB elsewhere said they only ever washed the membrane a few times in 0.1 % PBS tween or TBST and then reblocked with 5% non fat milk and then placed primary antibody for new marker.
In an ideal world all markers would be stained using just one membrane but i doubt this can be achieved.
Can anybody give me some advice on which would be the best way to progress if my proteins will be in a similar are to the beta actin as i dont want any residual bands to interfere with my markers.
Edited by cotchy, 18 August 2009 - 12:43 AM.