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Quantitative Western Imaging - (Sep/23/2010 )

Hi, I want to do some quantitative WB analysis. So I use the camera to take the pictures (chemiluminescent detection) for every 30sec. In that way, I get a series of pictures with different band density. I can tell by eyes that for the shorter exposure time, there is difference between two bands. However, for the longer exposure time, the difference is gone. I figure it should be due to the over exposure problem. So I am wondering if somebody has the experience dealing with this issue and can tell me how to choose the right picture without over exposure. Thank you so much!

-ninitutu-

It is called 'densitometric' analysis. I do it with NBT/BCIP developed membranes though, not chemilumnescent but the concept is the same. you need softwares that measure the density of light emission from areas on your membrane, then compare with your controls. This is relative quantification

-Curtis-

ninitutu on Fri Sep 24 05:12:16 2010 said:


Hi, I want to do some quantitative WB analysis. So I use the camera to take the pictures (chemiluminescent detection) for every 30sec. In that way, I get a series of pictures with different band density. I can tell by eyes that for the shorter exposure time, there is difference between two bands. However, for the longer exposure time, the difference is gone. I figure it should be due to the over exposure problem. So I am wondering if somebody has the experience dealing with this issue and can tell me how to choose the right picture without over exposure. Thank you so much!


I know Quantity One (BioRad) will do this type of analysis. If you know someone that has a Gel Doc you might ask if you can analyze your images with their software. As far as over exposure, you could check your standards and if your upper concentrations flatten out then you are overexposed. Maybe take a couple exposures and use one that gives you a good curve.

-MoleculeMan-

Thanks, guys. I am trying to find the software (from Biorad) to do that.

-ninitutu-