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IP quantification

IP quantification - Proteomics Forum

IP quantification - Post Questions and Discuss Proteomics, Proteomic Bioinformatics, Proteomic Techniques such as 2-D, Mass Spec etc.


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  #1  
Old 08-17-2009, 08:55 PM
Pipette Filler
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Talking 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.
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Old 08-18-2009, 05:22 PM
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Default 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.
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Old 03-26-2010, 06:58 PM
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Default Re: IP quantification

Quote:
Originally Posted by danfive View Post
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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