IP quantification

[ceciloo1]

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]

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]

Originally Posted by danfive

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.