Western blotting question - How to strip a mambrane and how many times can it be used (Aug/18/2009 )
Hi all,
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.
Thank you.
cotchy on Aug 18 2009, 12:41 AM said:
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.
Thank you.
I have used the twice stripped blots with good success, but there were times when I lost it even with one stripping! It does depend upon a lot of things, including the amount of protein that is there on the membrane to be detected, and the amount of protein (signal) to be stripped. And that is even if you discount the technical variation of protocols and hands that use them. So, I don't think there is any set answer to this question. I generally use pierce kit of western blot stripping.
If you want to look for many protocols at once and compare, see these protocols.
Thanks cell counter i will have a look at these.
cotchy on Aug 19 2009, 06:35 AM said:
Also, just to make life slightly easier, why not use a different marker than B-Actin, since your targets all run around that molecular weight? Use GAPDH for a lower MW or tubulin for a higher MW, or even RNA pol II for a much greater MW band. One less time that stripping would be needed...
Might be better if you use IR-dye secondary antibodies that show different colors? In my case, beta-actin expressed much stronger band compared to other proteins. So I would leave it to the last since it's harder to be stripped off.
Can you use PVDF instead? It holds up better to multiple strip and reprobes
Hey
if your proteins are in a close range can you run them on a gradient-gel? Also different species can help...if your primary Abs are from different species: you can first detect with anti-rabbit for example and then anti-mouse....
When I strip a blot, i use H2O2 1:1, to destroy the HRP, this doesn-t affect your protein (too much)
Maybe this can help?
Good luck
cotchy on Aug 18 2009, 10:41 AM said:
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.
Thank you.
fysio lab on Sep 14 2009, 05:29 AM said:
if your proteins are in a close range can you run them on a gradient-gel? Also different species can help...if your primary Abs are from different species: you can first detect with anti-rabbit for example and then anti-mouse....
When I strip a blot, i use H2O2 1:1, to destroy the HRP, this doesn-t affect your protein (too much)
Maybe this can help?
Good luck
For qualtitative purposes, i strip a PVDF membrane up to 3 times max and still i see a signal.However best is not to strip more than once.
cotchy on Aug 18 2009, 10:41 AM said:
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.
Thank you.