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Blocking buffer for phospho-(AKT, GSK3b) WB using Odyssey?

Odyssey buffer phospho

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#1 proteinrunner

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Posted 25 February 2013 - 02:13 PM

Hi,

I'm doing phospho-(AKT, GSK3b) WB using Odyssey blocking buffer:PBS 1:1but only see very week signals, and some unspecific bands. I wonder what is the optimal blocking buffer to use. The instruction provided by Licor shows a WB figure comparing Odyssey blocking buffer:PBS 1:1, 5% BSA, etc, but I don't see much difference there. On the other hand I heard that BSA is better than the Odyssey blocking buffer. Does anyone have experience on this? Which BSA concentration to use? (I'm using nitrocellulose membrane)

Thanks in advance.
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#2 bob1

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Posted 25 February 2013 - 02:21 PM

Basically, the only way to find out is to trial different buffers until you find one that works well. Not all antibodies work well in BSA/PBS and not all antibodies work in the Odyssey blocking buffer either, though most will work in both. You can try adding some tween-20 (0.1%) to the block. Note that you should titrate the primary and secondary antibody dilutions to reduce non-specific bands, don't just rely on the dilution recommended by the company!

If you are using the fluorescent odyssey capabilities, the membrane type you use can have a big effect. It is best to get a fluorescence rated one to ensure that the background is minimized.





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