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colorimetry vs chemiluminescence


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#1 josephine

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Posted 09 July 2009 - 11:50 PM

Hi everybody!

I have always been using the HRP chemiluminescent method to develop my membranes. However, in my new lab we don't have the equipment necessary so I thought about switching to AP colorimetric method. Has anyone had any experience with it? How much less sensitive is it? If I use ~10 g of protein per lane using the standard method, how much would I have to load to get comparable intensity using AP (NBT/BCIP as substrates)

Thanks for help!

j.

#2 Nrelo

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Posted 10 July 2009 - 09:34 PM

Hi everybody!

I have always been using the HRP chemiluminescent method to develop my membranes. However, in my new lab we don't have the equipment necessary so I thought about switching to AP colorimetric method. Has anyone had any experience with it? How much less sensitive is it? If I use ~10 g of protein per lane using the standard method, how much would I have to load to get comparable intensity using AP (NBT/BCIP as substrates)

Thanks for help!

j.


AP is less sensitive than HRP. I think you have to optimize your protocol when using AP. In my protocols, I usually use 1~5ug per lane. One shortcome of AP is that the membrane cannot be washed after developing. In HRP, you can wash the membrane to remove reagents and detect the bands again.

I have switched from AP to HRP. In the past I found that AP always give you pink or purple background. If you target is hard to be detected. This could be a problem.




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