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ceciloo1 08-17-2009 08:55 PM

IP quantification
 
Hi,

I'm student and I've done an immunoprecipitation followed by western blot. I would like to know if it's possible to quantify this IP-western despite we don't know exactly the amount of immunoprecipitated proteins? maybe with the relative intensity of band (compared to my positive control)?

thx,
cecile.

danfive 08-18-2009 05:22 PM

Re: IP quantification
 
The WB can be used to predict concentrations of unknowns/samples as well.
I use Quantity One software--I set up stds in lanes (~3; high, med, low conc'ns)--and the software allows me to circle the bands and input the known concentrations. Then, voila, I have a linear std curve. That then predicts the conc. of unknown samples.

What it does is measure intensity/mm^2.

Of course I have to check the accuracy of this software:
on gel bands (coomasie blue stain)--accuracy, measured in % error 3.4-11.25%
on Western Blot (luminiscent signal)--accuracy, measured in % error 4.67-13.1%

Not too, shabby.

mehcp 03-26-2010 06:58 PM

Re: IP quantification
 
Quote:

Originally Posted by danfive (Post 410488)
The WB can be used to predict concentrations of unknowns/samples as well.
I use Quantity One software--I set up stds in lanes (~3; high, med, low conc'ns)--and the software allows me to circle the bands and input the known concentrations. Then, voila, I have a linear std curve. That then predicts the conc. of unknown samples.

What it does is measure intensity/mm^2.

Of course I have to check the accuracy of this software:
on gel bands (coomasie blue stain)--accuracy, measured in % error 3.4-11.25%
on Western Blot (luminiscent signal)--accuracy, measured in % error 4.67-13.1%

Not too, shabby.

I've successfully used similar protocols.


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