Denaturing Reagent for Western Blot - (May/14/2010 )
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...
HomeBrew on May 19 2010, 09:44 AM said:
i second that!!
HomeBrew on May 18 2010, 07:14 PM said:
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).
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?
chicho on May 20 2010, 04:39 AM said:
HomeBrew on May 18 2010, 07:14 PM said:
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)
Well, I have some further questions, which is why I wanted to see if I understood the situation accurately. Let's say, until chicho gets back, that the situation as I posted it above is accurate...
I can see how one might wind up with two fractions containing the same protein after size exclusion if there were two forms of the same protein put through the column. What I'm not clear on is that if these two fractions each produce the same sized band on SDS-PAGE, is that not evidence that, under the SDS-PAGE conditions, the proteins are now equally denatured (i.e. in the same form)? If they were still in two different three dimensional forms, wouldn't they migrate differently through the gel?
And if they're now in the same form, as evidenced by them producing the same sized band on SDS-PAGE, why do they react differently, at least in degree of intensity, on western blot?
Other scenarios are possible -- the two forms of protein differ from one another by some post-translational modification, like the cleavage of a signal sequence, or by attachment of some functional group like acetate, phosphate, or carbohydrates, or some other post-translational change, although it would have to be an alteration that does not result in a change to the apparent molecular weight on SDS-PAGE.
If this is the case, you can't really say they are the same protein -- you'd have to say one is 'nascent' and the other 'mature' or something. And, if these proteins differ by some such change, then reactivity on western could certainly be altered without changes to the intensity of Coomassie staining, with either a monoclonal or polyclonal sera.
Or, there could be paralogous genes in whatever organism this is that produce slightly different versions of the same protein. This could also result in the experimental observations made.
Or (probably least likely), one of the fractions could be a different protein altogether, but one with properties similar to the desired protein such that it elutes off a column at about the same time, and shows some limited cross-reactivity with the sera used on western blot.
I'm not necessarily agreeing with "reviewer-prick#1" -- I don't have enough information to say whether there's a realistic chance that the two fractions don't in fact contain the same protein. But, since there are at least several possible scenarios that would result in the observations reported, maybe some further bullet-proofing is needed...
Maybe one could do something like comparing the patterns produced by digestion with trypsin or some other protease, or do some protein sequencing or mass spec work to show the proteins in the fractions are in fact the same.
HomeBrew on May 20 2010, 06:24 AM said:
I can see how one might wind up with two fractions containing the same protein after size exclusion if there were two forms of the same protein put through the column. What I'm not clear on is that if these two fractions each produce the same sized band on SDS-PAGE, is that not evidence that, under the SDS-PAGE conditions, the proteins are now equally denatured (i.e. in the same form)? If they were still in two different three dimensional forms, wouldn't they migrate differently through the gel?
And if they're now in the same form, as evidenced by them producing the same sized band on SDS-PAGE, why do they react differently, at least in degree of intensity, on western blot?
Other scenarios are possible -- the two forms of protein differ from one another by some post-translational modification, like the cleavage of a signal sequence, or by attachment of some functional group like acetate, phosphate, or carbohydrates, or some other post-translational change, although it would have to be an alteration that does not result in a change to the apparent molecular weight on SDS-PAGE.
If this is the case, you can't really say they are the same protein -- you'd have to say one is 'nascent' and the other 'mature' or something. And, if these proteins differ by some such change, then reactivity on western could certainly be altered without changes to the intensity of Coomassie staining, with either a monoclonal or polyclonal sera.
Or, there could be paralogous genes in whatever organism this is that produce slightly different versions of the same protein. This could also result in the experimental observations made.
Or (probably least likely), one of the fractions could be a different protein altogether, but one with properties similar to the desired protein such that it elutes off a column at about the same time, and shows some limited cross-reactivity with the sera used on western blot.
I'm not necessarily agreeing with "reviewer-prick#1" -- I don't have enough information to say whether there's a realistic chance that the two fractions don't in fact contain the same protein. But, since there are at least several possible scenarios that would result in the observations reported, maybe some further bullet-proofing is needed...
Maybe one could do something like comparing the patterns produced by digestion with trypsin or some other protease, or do some protein sequencing or mass spec work to show the proteins in the fractions are in fact the same.
So here is more info:
I am expressing a protein in E. Coli. My total protein sample is coming from the inclusion body of the bacteria. If I run this sample on SDS PAGE and stain with comassie, then I get very clean band at the MW expected. Now take this product after refolding and run it through size exclusion and test all fractions separately. Basically the two samples I have been taking about are actually two different fractions of my total sample. The thing about my protein is that it is known to adopt different aggregation states and also different secondary structures (and most likely tertiary too). I cannot assume that an aggregated is composed of monomers of a single type of secondary structure (and most likely tertiary too). It has also been reported that SDS + heat does not completely denature this protein as opposed to guanidinium.
I hope this makes things a little more clear (I guess it could also do the opposite - make things even more complicated).