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protein size gradient gel


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10 replies to this topic

#1 torte

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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

#2 T C

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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

#3 Sciurus

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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.

#4 torte

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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.

#5 torte

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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.

#6 Sciurus

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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...

#7 torte

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Posted 31 March 2009 - 12:51 AM

yes, concentration gradient, sorry :)

#8 Sciurus

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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.

#9 mdfenko

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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.
talent does what it can
genius does what it must
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#10 torte

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Posted 02 April 2009 - 10:14 PM

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

#11 mdfenko

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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.
talent does what it can
genius does what it must
i do what i get paid to do




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