help with interpreting protein SDS analysis

[SmoothOperator]

First part:

 

1) Used SDS page - size of protein: 4kilodalton

2) Used SDS page again - this time using reagents to break disulphide bonds - still 4kilodalton

 

--> What does this mean? why are the results the same?

 

--> how many peptide strands should i expect given 36 amino acids from the first SDS- page, which assumed 110 daltons/amino acid (4000/110 ~ 36)

 

Second part:

 

protein was analyzed: amino acid sequence as follows:

A^2 C^2 D^2 E^4 F G^3 H L M N^2 P^2 Q^2 R^3 S^4 T^3 W, dansylated-R, and dansylated-K

 

does this make sense given results from the first part??

 

Is it possible for two amino acids to be dansylated (I thought there was only 1 N-terminus amino acid?)

 

Thanks- stumped here!

[Greippi]

If you're querying why it only runs as one band:

 

Correct me if I am wrong, but when you prepare protein for SDS PAGE, are you not merely denaturing the protein (i.e. causing it to lose its structure, but not breaking bonds between consecutive amino acids)? Of course, it depends on how you have prepared the protein before you load it on to the gel, but usually you would expect a protein to run as one band.

Breaking disulphide bonds merely breaks down the tertiary structure further and would not break the peptide bonds holding the chain of amino acids together, so it would still run as one band.

 

I think this may be your main issue though:

Your peptide is very small. SDS PAGE is not generally used for the analysis of peptides. Very specialised systems are available for larger peptides (greater than 50 residues) , but on a normal SDS gel, the peptides just run at the buffer front - your size of 4kD may just have been the buffer front! Other problems with peptides include poor staining and the fact that they don't fix in the gel.

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Merged post follows:

Consecutive posts merged

As for your "protein was analysed", what methods were used? The numerical values, are they how many residues actually are in the peptide, or are they the molar ratio results of an amino acid analysis?

Once I know this, I can help you a bit more.