Gel filtration chromatography

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Hi,
I am studying for my biochemistry lab exam. While going over one of the questions... I came across one that I can't answer...help will be appreciated.

Question under analysis is related to a previous question.

(Previous question)
A gel filtration (size exlcusion) is set up and the following monomeric proteins are separated
The buffer used was: 20mM Tris-HCl, pH 7.0, 150 mM NaCl.
Protein A = 6 KDa
Protein B = 22 KDa
Protein C = 190 KDa
Protein D = 80 kDa

Predict the order of elution.


I understand that in a size exclusion chromatography, larger molecules elute first.. hence the order would be:

C, D, B, A.

(Question under analysis).
In the previous question a certain protein X ( M.W. = 20 kDa) eluted at about the time as protein C... what does this tell you about protein X?

I don't know what would this tell me about protein X... does it have something to do with the fact that protein X may be able to form dimers or polymers? (I.e. Hemoglobin with 4 different subunits, a dimer of αβ-protomer?

Thanks in advance
 
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  • #2
Ygggdrasil
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Hi,
I am studying for my biochemistry lab exam. While going over one of the questions... I came across one that I can't answer...help will be appreciated.

Question under analysis is related to a previous question.

(Previous question)
A gel filtration (size exlcusion) is set up and the following monomeric proteins are separated
The buffer used was: 20mM Tris-HCl, pH 7.0, 150 mM NaCl.
Protein A = 6 KDa
Protein B = 22 KDa
Protein C = 190 KDa
Protein D = 80 kDa

Predict the order of elution.


I understand that in a size exclusion chromatography, larger molecules elute first.. hence the order would be:

C, D, B, A.

Yes, that's correct.

(Question under analysis).
In the previous question a certain protein X ( M.W. = 20 kDa) eluted at about the time as protein C... what does this tell you about protein X?

I don't know what would this tell me about protein X... does it have something to do with the fact that protein X may be able to form dimers or polymers? (I.e. Hemoglobin with 4 different subunits, a dimer of αβ-protomer?

Thanks in advance

Yes, you are on the right track. You should also be able to say something the approximate number of subunits per oligomer based on the data (for example, is the protein likely a dimer?).
 
  • #3
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Just another hint to keep in mind along with Ygggdrasil's comment is that gel filtration columns are typical run under native, that is non-denaturing, conditions. Compare the conditions of size exclusion with those of SDS-PAGE for the determination of approximate molecular weights. Can you envision a situation where the two methods can give different numbers but still be in agreement with each other?
 

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