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protein size gradient gel - (Mar/30/2009 )

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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 donīt think this is appropriate for gradient gels. Thanks

-torte-

Hey,

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

TC

-T C-

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.

-Sciurus-

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

-torte-

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.

-torte-

So your gradient is PAA concentration?

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

-Sciurus-

yes, concentration gradient, sorry :)

-torte-

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

yes, concentration gradient, sorry :)


Ok, misunderstanding cleared up :D

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.

-Sciurus-

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.

-mdfenko-

Well, Iīve tried this using the most upper ladder band as unknown, but it doesnīt work.

-torte-
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