Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Western blotting question


  • Please log in to reply
7 replies to this topic

#1 cotchy

cotchy

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 87 posts
1
Neutral

Posted 18 August 2009 - 12:41 AM

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.

Edited by cotchy, 18 August 2009 - 12:43 AM.


#2 cellcounter

cellcounter

    Control Panel

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 145 posts
3
Neutral

Posted 18 August 2009 - 08:29 AM

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.

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.

#3 cotchy

cotchy

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 87 posts
1
Neutral

Posted 19 August 2009 - 02:35 AM

Thanks cell counter i will have a look at these.

#4 Dr Teeth

Dr Teeth

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 221 posts
1
Neutral

Posted 19 August 2009 - 05:18 AM

Thanks cell counter i will have a look at these.


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...

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#5 jinx1987

jinx1987

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 19 August 2009 - 12:57 PM

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.

#6 miBunny

miBunny

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 130 posts
1
Neutral

Posted 19 August 2009 - 05:07 PM

Can you use PVDF instead? It holds up better to multiple strip and reprobes

#7 fysio lab

fysio lab

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 40 posts
0
Neutral

Posted 14 September 2009 - 04:29 AM

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





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.



#8 Julie123

Julie123

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 14 April 2010 - 08:49 PM

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
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.




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.






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.