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
- - - - -

Denaturing Reagent for Western Blot


  • Please log in to reply
16 replies to this topic

#1 chicho

chicho

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 14 May 2010 - 08:49 AM

If I have a protein that is not being fully denatured by SDS + heat, what denaturing treatment would be possible to perform so that I could still run the sample in a western blot?

#2 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 14 May 2010 - 07:32 PM

If I have a protein that is not being fully denatured by SDS + heat, what denaturing treatment would be possible to perform so that I could still run the sample in a western blot?


why dont u try GuHCl or urea!!
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#3 lab rat

lab rat

    Why does a science forum not have pictures of mice and rats?

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 245 posts
7
Neutral

Posted 15 May 2010 - 11:25 AM

Does your loading buffer contain beta-mercapo?
42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.

#4 chicho

chicho

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 17 May 2010 - 06:44 AM

If I have a protein that is not being fully denatured by SDS + heat, what denaturing treatment would be possible to perform so that I could still run the sample in a western blot?


why dont u try GuHCl or urea!!


Can I actually treat with guanidinium and then run SDS PAGE? I guess I would have to treat the proteins with 6M guanidinium and then dilute out the sample? How much would I have to dilute the sample? Then just add SDS loading buffer and continue as I would normally?

As far as urea goes, I was under the impression that having urea in the loading buffer and then heating the protein shouldn't be done.

#5 chicho

chicho

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 17 May 2010 - 06:47 AM

Does your loading buffer contain beta-mercapo?



Yes, it does.

#6 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
15
Good

Posted 17 May 2010 - 05:40 PM

So, boiling with BME and running through an SDS-PAGE gel does not denature your protein? Why did you conclude that your protein is not fully denatured?

#7 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 17 May 2010 - 08:04 PM

If I have a protein that is not being fully denatured by SDS + heat, what denaturing treatment would be possible to perform so that I could still run the sample in a western blot?


why dont u try GuHCl or urea!!


Can I actually treat with guanidinium and then run SDS PAGE? I guess I would have to treat the proteins with 6M guanidinium and then dilute out the sample? How much would I have to dilute the sample? Then just add SDS loading buffer and continue as I would normally?

As far as urea goes, I was under the impression that having urea in the loading buffer and then heating the protein shouldn't be done.


well they both will unfold your protein and if you are using a non-reduced buffer it is not necessary to heat... heating is done to facilitate the linearization as far as the non-reducing buffer is concerned which will be done by GuHCl or urea!!! and yes you can directly add the buffer after adding GuHCl by diluting and then run the gel!!
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#8 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 17 May 2010 - 08:05 PM

So, boiling with BME and running through an SDS-PAGE gel does not denature your protein? Why did you conclude that your protein is not fully denatured?


also the question here is what is your application? why do you wanna denature it at the first place??!! if u just wanna prove identity... is denaturing necessary!!!??!! :P
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#9 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,711 posts
123
Excellent

Posted 18 May 2010 - 10:50 AM

also, if your protein is partially denaturing then you may want to try heating to a lower temperature (60-70C) and incubate longer to ensure full denaturation.

by the way, how do you know it is partially denaturing (similar to homebrew's question)?
talent does what it can
genius does what it must
i do what i get paid to do

#10 chicho

chicho

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 18 May 2010 - 06:30 PM

also, if your protein is partially denaturing then you may want to try heating to a lower temperature (60-70C) and incubate longer to ensure full denaturation.

by the way, how do you know it is partially denaturing (similar to homebrew's question)?


Well to make a long story short: I have two fractions from size exclusion of the same protein. In other words, one fraction is more aggregated than the other. Runing SDS PAGE followed by comassie blue reveals both fractions at the same level. But running a western blot reveals that one of the fractions is many times more than the other (disagrees with the comassie staining). It has been reported previously that my protein is not fully denatured by SDS + heat. Thus, this lead me to test the possibility that the fraction with the least signal in western might not be fully denatured and the antibody is not gaining access to the epitope.

#11 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
15
Good

Posted 18 May 2010 - 07:14 PM

I would believe a quantitative estimate based on a Coomassie-stained gel more so than one based on a western blot -- one is telling you how much protein there is, and the other is telling you how many intact and accessible epitopes there are on those proteins...

#12 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 18 May 2010 - 07:52 PM

I would believe a quantitative estimate based on a Coomassie-stained gel more so than one based on a western blot -- one is telling you how much protein there is, and the other is telling you how many intact and accessible epitopes there are on those proteins...



i second that!!
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#13 chicho

chicho

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 19 May 2010 - 03:09 PM

I would believe a quantitative estimate based on a Coomassie-stained gel more so than one based on a western blot -- one is telling you how much protein there is, and the other is telling you how many intact and accessible epitopes there are on those proteins...


Ok, but here is where it gets tricky:

Lets say that there is this person criticizing my results, lets call him "reviewer-prick#1". Now, reviewer-prick#1 claims that the fraction that has a low western blot signal really has as much of my protein of interest as the western blot shows. He also claims that in my purification process I have somehow managed to co-purify some random artifact protein with the same MW as my protein of interest. Thus, I have to design experiments to prove reviewer-prick#1 wrong (or right).

#14 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
15
Good

Posted 20 May 2010 - 03:10 AM

Let me make sure I understand the situation accurately...

You ran size exclusion chromotography on a sample of some kind and obtained two fractions. These fractions, though they eluted at different times from the column, each produce a single band on a Coomassie-stained SDS-PAGE gel, and each of these bands is the same with regards to apparent molecular weight on the gel. Moreover, each of these two bands appears with the same intensity when stained with Coomassie, thus suggesting they contain equal amounts of protein.

However, if you run samples of these two fractions on an SDS-PAGE gel and transfer the gel to a membrane and perform a western blot, you get the same bands as regards equality of apparent molecular weight, but the sample from one fraction produces a more intense band, thus suggesting they may not contain equal amounts of protein.

Your thesis is that the protein contained in each of the two column fractions is in fact identical, and the reason that the protein eluted in two fractions rather than one is that there were two forms of the protein on the size exclusion column, differing from one another in three-dimensional conformation or degree of denaturation.

Your problem is that some reviewer doesn't believe you've adequately demonstrated that the protein contained in each of the two fractions is in fact the same.

Is this summation accurate?

#15 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 20 May 2010 - 03:35 AM

I would believe a quantitative estimate based on a Coomassie-stained gel more so than one based on a western blot -- one is telling you how much protein there is, and the other is telling you how many intact and accessible epitopes there are on those proteins...


Ok, but here is where it gets tricky:

Lets say that there is this person criticizing my results, lets call him "reviewer-prick#1". Now, reviewer-prick#1 claims that the fraction that has a low western blot signal really has as much of my protein of interest as the western blot shows. He also claims that in my purification process I have somehow managed to co-purify some random artifact protein with the same MW as my protein of interest. Thus, I have to design experiments to prove reviewer-prick#1 wrong (or right).



but if your western blot is specific then there is no question of a co-purification of sum unrelated protein!!! the one option that remains as hb also says is that sum modified form of your protein might have been generated which is giving different peaks (hydrodynamic radius different)
Support bacteria - They are the only culture some people have!!!
Cheers!!!




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

©1999-2013 Protocol Online, All rights reserved.