protein size gradient gel (View forum version)



torte

Posted 30 March 2009 - 10:04 PM

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

T C

Posted 30 March 2009 - 11:31 PM

Hey,

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

TC

Sciurus

Posted 30 March 2009 - 11:55 PM

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.

torte

Posted 31 March 2009 - 12:05 AM

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

Posted 31 March 2009 - 12:07 AM

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.

Sciurus

Posted 31 March 2009 - 12:32 AM

So your gradient is PAA concentration?

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

torte

Posted 31 March 2009 - 12:51 AM

yes, concentration gradient, sorry :)

Sciurus

Posted 31 March 2009 - 01:05 AM

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.

mdfenko

Posted 31 March 2009 - 07:10 AM

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.

torte

Posted 02 April 2009 - 10:14 PM

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

mdfenko

Posted 06 April 2009 - 10:38 AM

does your protein fall within the range of the standards?

if so, then you should be able to accurately extrapolate the mw from the curve.