protein size - (Oct/07/2010 )
Hi all, I have a trouble with the size of my protein. My protein is 430aa and has a 6*his tag at the N-terminal. So the total size is around 45-48kd. However, when I ran it in SDS-Page, the protein ran to ~70kd which is much bigger than the size calculated. Could someone give my some clues why it happens? Thanks so much! BTW, the protein is from Ecoli expression.
sds-page will give you an approximation of the molecular weight of a protein. this approximation can deviate from the actual molecular weight due to a number of factors.
one factor may be uneven distribution of sds on the protein. this will cause the charge density to be different from expected and will cause the protein to migrate at a different speed than other proteins. i suspect that this is the cause of your confusion.
to get a better estimate you can run your protein on gel filtration liquid chromatography.
to get the actual molecular weight you can use lc-ms.