What Does Protein X's Elution Time Suggest About Its Structure?

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SUMMARY

The discussion focuses on the elution behavior of protein X during size exclusion chromatography, indicating that protein X, with a molecular weight of 20 kDa, eluted at a similar time as protein C (190 kDa). This suggests that protein X may exist as a dimer or oligomer, affecting its elution profile. The participants emphasize the importance of understanding the implications of oligomerization in interpreting elution times and the differences between size exclusion chromatography and SDS-PAGE in determining molecular weights.

PREREQUISITES
  • Understanding of size exclusion chromatography principles
  • Knowledge of protein oligomerization and its effects on molecular weight
  • Familiarity with buffer compositions, specifically 20mM Tris-HCl, pH 7.0, 150 mM NaCl
  • Basic concepts of SDS-PAGE and its comparison to size exclusion chromatography
NEXT STEPS
  • Research the principles of size exclusion chromatography and its applications in protein analysis
  • Learn about protein oligomerization and methods to determine oligomeric states
  • Study the differences between size exclusion chromatography and SDS-PAGE for molecular weight determination
  • Explore case studies of proteins that exhibit dimerization or polymerization in gel filtration settings
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Biochemistry students, researchers studying protein structure, and laboratory technicians involved in protein purification and analysis will benefit from this discussion.

hyddro
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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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hyddro said:
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?).
 
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?