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

In summary, the conversation discusses a question related to a previous question about gel filtration and the separation of monomeric proteins. The predicted order of elution is C, D, B, A due to size exclusion. The question under analysis asks about a protein X that eluted at the same time as protein C and what this could indicate about protein X, potentially its ability to form dimers or polymers. The conversation also mentions the use of native conditions in gel filtration and the differences between this method and SDS-PAGE for determining molecular weights.
  • #1
hyddro
74
2
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
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?).
 
  • #3
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?
 

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

What is gel filtration chromatography?

Gel filtration chromatography, also known as size exclusion chromatography, is a type of liquid chromatography used to separate molecules based on their size. It works by passing a mixture of molecules through a column filled with porous beads, which act as a sieve to separate larger molecules from smaller ones.

What is the principle behind gel filtration chromatography?

The principle behind gel filtration chromatography is that smaller molecules will take longer to travel through the column because they can enter the pores of the beads, while larger molecules will travel faster because they are excluded from entering the pores. This results in separation of the molecules based on their size.

What are the advantages of using gel filtration chromatography?

Gel filtration chromatography has several advantages, including its ability to separate molecules based on size without the need for specialized equipment, its ability to handle a wide range of sample sizes, and its gentle nature which allows for the separation of fragile molecules.

What are the limitations of gel filtration chromatography?

One limitation of gel filtration chromatography is that it is not suitable for separating molecules with similar sizes. It also has a lower resolution compared to other chromatography techniques, and may not be able to separate molecules with very large or very small sizes effectively.

How is gel filtration chromatography used in research?

Gel filtration chromatography is commonly used in research to purify and analyze biological molecules such as proteins, nucleic acids, and carbohydrates. It is also used for quality control and to determine the molecular weight of unknown compounds. In addition, it can be used to remove unwanted molecules from a sample and to concentrate dilute samples.

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