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How is the mass detected via SDS-PAGE?


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#1 zabbn

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Posted 11 November 2013 - 05:56 AM

Hello,

 

after some research it still remains unclear to me, how a mass-dependent separation of proteins is possible, if using SDS-PAGE. I have read, that one molecule SDS binds to a protein every two aminoacids. That would not provide a charge proportional to mass, as different aminoacids have different molecular weights. Often it is said, that SDS binds in a ratio of 1,4 g SDS / 1 g protein. That would garantuee a charge in proportion to the weight of the protein. But how is that accomplished by the SDS molecule? And how is that possible, if SDS binds to a protein every two AA? And what does that mean for phosphorylated proteins? They should be heavier, but is that even recognized in the amount of negative charges attached by SDS?

 

Thank you for answers!

 

Best regards,

-zabbn-

 

PS: apologies for my english



#2 jerryshelly1

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Posted 11 November 2013 - 07:36 AM

You are correct, but I think you are over analyzing SDS-PAGE. SDS binds quantitatively to proteins, with one SDS molecule binding to ~2 amino acids. SDS is super negative, meaning that any charge the protein has is overcome by SDS. This allows the protein to separated solely of size. The heaver the protein (kilodalton), the slower it will migrate through the mesh work of the gel. SDS has a tail that binds to and linearizes the overall structure of the protein (link below).

 

Phosphorylated proteins are detected in the same manner. If you run a blot and you see multiple banding patterns above your predicted size, it is a good indication that the protein has undergone some type of post-translational modification. Antibodies can be developed to specifically recognize whether a protein is phosphorylated (if that helps).

 

General WB Information:

http://www.bio-rad.c...horesis-methods



#3 zabbn

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Posted 12 November 2013 - 03:20 AM

Thank you for your answer,

but that doesn't quite explain to me, why a protein, that is linearized and negatively charged at any AA, will move with different speed through a mesh, if it has just the same size but a different mass (Da). Because any protein that consists of the same number of amino acids but different types of amino acids will have a different weight but "look" the same if linearized and negatively charged.

To the phosphorylation problem: There are people who claim, that an identical protein, that is present in e.g. two phosphorylation patterns will result in two bands on the gel.

So my question is: How does the separation on the gel happen by weight, if every protein will be linearized and negatively charged in the same way? Why does a protein consisting of e.g. 30 tryptophan run slower than a protein consisting also of 30 amino acids, but all of them alanine?



#4 jerryshelly1

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Posted 12 November 2013 - 07:47 AM

Even if two proteins have the same number of amino acids, there relative masses will be different. Look at the structure of tryptophan vs. glycine. It is all about composition. Even though two proteins have the same number of amino acids DOES NOT mean that they will have the same size.

 

The phosphorylation state of a protein adds additional mass, causing the protein to migrate slower through the gel.

 

Again, tryptophan has a greater mass than alanine and will cause the protein to run slower through the gel.

 

Is this alright?






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