protein size gradient gel - (Mar/31/2009 )

Hi,
does anyone knows, how to calculate the size of a protein band when using gradient gel? I know how to do this when using "normal" gels, but I don´t think this is appropriate for gradient gels. Thanks

Hey,

NO experience with gradient gels but won't running a standard ladder under the same conditions help?

TC

I don't know if it's even possible to calculate band size from a gradient gel because you don't separate based on size but rather based on sequence differences that denature differently.
A marker won't help either because it would also denature at specific sequences and not run according to size.

My suggestion would be to run the sample on a normal gel and calculate from that.

why it is not possible to calculate the size? It denatures the same way as the non-gradient gel, doesn´t it? It is 4-15% PAAG gel. I can´t run it on non-gradient gel, because I need to see proteins of all sizes.

And of course the ladder migrates the same way as my protein, but I need to know the size of protein inbetween to ladder bands.

So your gradient is PAA concentration?

I thought that you meant temperature or some other denaturing agent gradient...

yes, concentration gradient, sorry

torte on Mar 31 2009, 10:51 AM said:

Ok, misunderstanding cleared up

Then as long as the marker is running on the same concentration as your sample you can calculate the band size like you'd normally do.
Maybe there is a way to include the different concentrations in the calculation - but I don't know of any, sorry.

just as with a single concentration gel, run the standards, plot a standard curve (mw vs. migration (or rf)), and see where your protein lands on the curve.

Well, I´ve tried this using the most upper ladder band as unknown, but it doesn´t work.