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RIPA Buffer precipitation trouble


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#1 A0110

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Posted 05 March 2010 - 12:28 PM

I am using the following composition of RIPA buffer: 1X RIPA (1% DOC Na+, 1% TritonX-100, 0.1% SDS, 1M Tris-Cl, pH 7.4). The buffer is precipitating few days after I add the protease inhibitors?

Does any one have an idea what could cause this?

Thanks

#2 laurequillo

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Posted 05 March 2010 - 12:47 PM

I am using the following composition of RIPA buffer: 1X RIPA (1% DOC Na+, 1% TritonX-100, 0.1% SDS, 1M Tris-Cl, pH 7.4). The buffer is precipitating few days after I add the protease inhibitors?

Does any one have an idea what could cause this?

Thanks


Which inhibitors? and why dont you add them just before use it?
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#3 A0110

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Posted 06 March 2010 - 02:02 PM

I am using the Protease Inhibitor Cocktail "Complete". I tried today to add only iodacetamide to RIPA buffer stock and will be adding the protease inhibitor tablets just before use.

#4 lab rat

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Posted 06 March 2010 - 08:19 PM

Are you using the EDTA+ or -free complete? (just a random guess, the EDTA may be precipitating out?)

Edited by lab rat, 06 March 2010 - 08:21 PM.

42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.

#5 bob1

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Posted 07 March 2010 - 03:44 PM

be aware that protease inhibitors only function for a few days after being put into solution (and only last approx 6 months frozen in solution).

#6 A0110

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Posted 10 March 2010 - 11:51 AM

I was using EDTA free RIPA. But I knew what was causing my problem. I used 1M Tris buffer to make up 1X RIPA. I should have used a final concentration of 0.1 Tris HCl. So, that should have been precipitating my buffer once I add anything to it.

Thank you guys.




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