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Do you get insoluble proteins also in whole cell lysate?


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3 replies to this topic

#1 neuron

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Posted 21 December 2010 - 11:48 PM

Hi,

I have this general question. When you lyse the whole cell pellet with any lysis buffer containing detergents like NP40 etc., do you get insoluble proteins also??? Because in general protocols we add lysis buffer in the pellet, keep it at ice for some time and then spin to collect the supernatant. Eventually we throw the pellet.What if you are looking for some specific protein and that goes in the pellet :( ...I may be wrong coz we are adding detergents to disrupt the membrane. But just wanted to check with people here on the forum. Hope somebody would be able to understand :)

Thanks

#2 mdfenko

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Posted 22 December 2010 - 08:54 AM

some proteins which may be insoluble, normally, will solubilize with detergents.
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#3 SonjaK

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Posted 30 December 2010 - 02:04 PM

It is possible to get proteins trapped in membranes etc to solubilize better by using detergents but keep in mind that if the protein you are looking for is an enzyme the activity of the protein may be lost. I would try out different detergents; SDS (this is strong), triton X-100 and CHAPS (these are more gentle ones) and see which one gives the best results.

#4 MyProteinBulliedMe

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Posted 31 December 2010 - 05:45 AM

I usually put 0.5% Triton X-100 and this is enough to solubilize most protein that I've purified. I've never use SDS because it's too strong. NP40 is 2-3x more stringent than Triton so I guess it depends on your protein/activity.




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