Native molecular weight

[ridhima]

My protein molecular weight is coming 40.7 kDa by gel-filtration and by gradient native page it is coming near 146 kDa and by SDS-PAGE it is coming near 40 kDa. I am not understanding this result as by gel- filtration and native PAGE molecular weight should come approximately same.

[Maximilian]

Is your protein a tetramer?

 

If the monomeric unit is 36.5kD that could explain the results.

Edited August 11, 2013 by Maximilian

[ridhima]

My protein is eluting at 80 ml in gel filtration. How can we say it is tetramer or monomer and how are you comparing this with SDS-PAGE results? Please explain to me in detail?

[Maximilian]

How are you running your gel filtration? If there are no denaturing agents and the pH is right, it should look the same as native.

 

In native, you are keeping the proteins in their native form. This means that the quaternary structure should remain conserved. On the SDS-PAGE you use denaturant (SDS to disrupt secondary/tertiary and a reducing agent such as beta-mercaptoethanol to reduce the disulfide bridges) to denature your protein. This will convert multimers into their monomeric form.

 

When you run gel filtration you can choose your conditions to keep the protein native or you can also denature it. If your conditions are denaturing, the results will likely look like SDS-PAGE. Otherwise they should look like native.

 

I am thinking your protein is a homotetramer because the small weight is approximately 1/4 of the bigger weight, but this is not necessary.

If you are running the SDS-PAGE and the native after purifying the protein with a his-tag, and there is only one band in the SDS-PAGE, I would think it is a homotetramer. It is hard to know without knowing exactly what the protein has gone through and your gel filtration conditions.

[ridhima]

Thanks Sir for your solution. Now I am clear. I have added DTT in buffer used for gel filtration that's why protein's molecular weight from SDS-PAGE and gel filtration is approx. 40 kDa and from Native Page it is coming approx. 146 kDa i.e. actually it is homotetramer.

[Maximilian]

Thanks Sir for your solution. Now I am clear. I have added DTT in buffer used for gel filtration that's why protein's molecular weight from SDS-PAGE and gel filtration is approx. 40 kDa and from Native Page it is coming approx. 146 kDa i.e. actually it is homotetramer.

 

Yup, that seems about what you'd expect under those conditions! I'm glad I could help.

[ridhima]

ya thanks a lot in solving my problem