Lysing cells with RIPA containing SDS - Stickiness of the lysate (Aug/22/2008 )

When I lyse the cells with RIPA that contains SDS, cell lysate is kinda of sticky, because the nucleus is broken and DNA is released.

I was wondering if it is necessary to sonicate the lysate to break the stickness, if the lysate is used for western?

Thanks.

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I sonicate to break up lysed matter and, really, because it will be easier to pipet into the gel.

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sometimes when you use RIPA you get clumps of cell lysate, if you carry out everything on ice and do less pipetting up and down you will eliminate this.

But, when my friends get clumps they add DNAse to their samples, and everything becomes ok immediately. you can try that. add DNAse to your clumped cell lysate. I've never tried this before, but my friends are happy with this procedure.

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That is good to know. Thanks.

In that case, I will try lysing without vigorously scraping the cells. Probably that could solve the problem.

As for DNAse, how much Dnase does your friend use?

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The problem is you are not clearing out the genomic DNA and this is what makes your sample sticky. Sonication only helps break up cells and shear the DNA and hence help the stickiness but it is not necessary. You should be spinning your lysates at maxRPM, 4degree for about 20-30 mins after lysis. You will get a pellet of DNA and now your samples are not sticky.

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I'm not sure, i'll try to ask if I don't forget. I think they used 10 ul. but I'm not sure.

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