Elcetrophoresis Question ( Protein Size) - (Nov/23/2006 )

Really appreciate if anyone could help:

1) Theproteins are seperated by elctrophoresis by the size. In exectrophoresis, the SDS unwinds the protein, and covers with negative charges. So the longer the protein, the greater the charge on it. Since mobilty of th protein is dependent on charge wont the longer proteins go much faster?

When the mean size are they meaning the length or the thickness?
anoop

-anoopbal-

QUOTE (anoopbal @ Nov 23 2006, 07:12 PM)

Nope. SDS covers the whole protein which makes proteins carry roughly the same charge density, thus overrides/cancels any effect due to charged residues of proteins on the migration rate. Under such a condition, the small pore size of PAGE gel imposes more resistance on large proteins than smaller ones. Thus the migration rate becomes proportional to MW.

-genehunter-1-

hi i can make it easier for you
we are trying to separate different proteins of different shapes and sizes right? so its better to denature them first so that the proteins no longer have any secondry , tertiary or quaternary structure they are now only primary amino acid structure and as SDS cover our proteins with the same negative charge so the protein got no other way to run through the gel pores than its size or MW.

-spanishflower-

Thanks for all the reply.

I understand about how SDS covers it with negative charges. My questions if the primary sequence is longer, wont there be more charges on it compared to smaller sequence? ANd sont the charge density effect the migration rate.

---------------- Longer protein with more negative charge

----- shorter protein with few negative charges

And if its just primary what is size being reffered to?

Hope you get what i am trying to convey

thanks
Anoop

-anoopbal-

If you run protein samples by electrophoresis in free solution (without any matrix, such as in capillary electrophoresis) the rate of migration of macromolecules is determined by charge/mass ratio. If SDS covers the whole molecule, particularly when all the disulfide bonds are disrupted, them all coated proteins now have roughly equal charge/mass ratio and will migrate approximately a same rate. The situation is completely different when gel matrix is used, in which molecules are separated based on combined effects of size and charge difference. Since we have largely override the charge effect using SDS coating, then the separation will be determined by mainly protein size differences.

-genehunter-1-

Thanks Genehunter. The charge to mass ratio makes sense now.

-anoopbal-