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Denaturing Reagent for Western Blot - (May/14/2010 )

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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?

-chicho-

chicho on May 14 2010, 11:19 PM said:

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!!

-Prep!-

Does your loading buffer contain beta-mercapo?

-lab rat-

Prep! on May 14 2010, 07:32 PM said:

chicho on May 14 2010, 11:19 PM said:

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.

-chicho-

lab rat on May 15 2010, 11:25 AM said:

Does your loading buffer contain beta-mercapo?



Yes, it does.

-chicho-

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?

-HomeBrew-

chicho on May 17 2010, 09:14 PM said:

Prep! on May 14 2010, 07:32 PM said:

chicho on May 14 2010, 11:19 PM said:

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!!

-Prep!-

HomeBrew on May 18 2010, 08:10 AM said:

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

-Prep!-

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)?

-mdfenko-

mdfenko on May 18 2010, 10:50 AM said:

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.

-chicho-
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