how to get rid of the unsoluble stuff in sheared lysates? - (Sep/10/2006 )

Hi,

Recently, I performed a series of ChIPs, after sonication, generally we will centrifuge the sample to spin down the unsoluble stuff. however, even we can spin down most of them, there is always something smoky like, hanging in the sheared lysates, I tried use full speed 20min, still can not get rid of them?

Does anyone have had the same problem? how can you deal with it?

Thank you.

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try higher centrifugation if possible


it's better to lysate your sample very well from the begining to avoid such smoky stuff..

by the way, what sample u are working on?!

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Hi strawberry,

Thank you for your reply, in fact, I even used more than 20000g for 20min, still can not get rid of them. By the way, what steps do you use to lysize the cells and then shear them by sonication? Thank you.

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You may need to use ultracentrifuge to get rid of all the particular stuff. 100,000gx30-60min.

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but I don't have this fancy one, any other idea? thank you.

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perhaps PEG fractionation ( using less amt for DNA precipitation)? You will have to play around with the concentration though.

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I met similar problem, and I agree that it is because you put in too many cells in small amount of lysis buffer. Reducing the cell numbers should work. Good luck!

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How many cells and which amount of buffer did you use?

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is it possible to use more buffer?
second though : pipett the solution, put in an other tube and spin again.
That worked for me in cell protein preparations.

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Hey,

The "smokey" stuff is actually lipids from the cells.
This lipid layer should be contained on the very top of your sample if you sppin at high speen for sufficient time.To avoid the lipids (they do not hurt the IP because they will
bind to beads during preclearing) use more buffer and put the
tip of your pipette through this layer and stuck up as much as you can without
taking up the lipids. The small amount you transfer will stick to the beads.

M___

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