SDS-PAGE running strangely - (Jun/17/2004 )
QUOTE (Brainzhang @ Jun 26 2006, 03:54 AM)
Hi everyone:
According to the molecular mass was overestimated or lessestimated on SDS-PAGE maybe that the special amino acid composition.
High content of hydrophobic residues possibly results in poor binding of SDS, and then run slower;
low content of hydrophobic residues possibly results in strong binding of SDS, and then run faster;
Following is the weblinker for hydrophobic content assay:
http://www.expasy.org/tools/protscale.html
Good Luck!
According to the molecular mass was overestimated or lessestimated on SDS-PAGE maybe that the special amino acid composition.
High content of hydrophobic residues possibly results in poor binding of SDS, and then run slower;
low content of hydrophobic residues possibly results in strong binding of SDS, and then run faster;
Following is the weblinker for hydrophobic content assay:
http://www.expasy.org/tools/protscale.html
Good Luck!
I am facing the same problem, I expressed the protein in E.Coli and use ExPasy to predicted MW of my his-tagged protein, it shows 41.5kD, while it appears to be ~35kD when I ran 10% SDS-PAGE gel (50volt for stack gel and 100volt for seprate gel), I am using pre-stained marker from Invitrogen. I did western blotting by his-tag Ab and it shows a band between 30-40kD. I am hisitated to take this band as my protein as the differences look very big. Is there anyone who has this kind of experiences? thanks.
-rabbit-
QUOTE (rabbit @ Jul 27 2006, 08:42 PM)
QUOTE (Brainzhang @ Jun 26 2006, 03:54 AM)
Hi everyone:
According to the molecular mass was overestimated or lessestimated on SDS-PAGE maybe that the special amino acid composition.
High content of hydrophobic residues possibly results in poor binding of SDS, and then run slower;
low content of hydrophobic residues possibly results in strong binding of SDS, and then run faster;
Following is the weblinker for hydrophobic content assay:
http://www.expasy.org/tools/protscale.html
Good Luck!
I am facing the same problem, I expressed the protein in E.Coli and use ExPasy to predicted MW of my his-tagged protein, it shows 41.5kD, while it appears to be ~35kD when I ran 10% SDS-PAGE gel (50volt for stack gel and 100volt for seprate gel), I am using pre-stained marker from Invitrogen. I did western blotting by his-tag Ab and it shows a band between 30-40kD. I am hisitated to take this band as my protein as the differences look very big. Is there anyone who has this kind of experiences? thanks.
MW determination by electrophoresis is not so precise.
Change your marker, and you will see another relative molecular weight.
Also, pre-stained molecular weight migrates differently depending on the pH (look for example the difference between tris-glycine and tri-tricine)
And you could also have some post-translation modification like glycosylation
-Missele-
Hi,
I have the same problem... my protein is 18kDa but t runs as a ~25kDa (tha same 7kDa dleta...).
I'm now trying to see wwather the protein is acetylated using Cell signaling Abs.
funny it's the same delta....
Lior.
-Lior_m-