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Insoluble material floats over my SDS lysate of human cells after centrifugation


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7 replies to this topic

#1 scddfmsku

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Posted 03 September 2012 - 05:14 AM

Hi!
I have a problem about very basic cell lysis. I lysed human cells (a cultured cell line) with SDS-tris buffer. The solution was cloudy, so I thought it was due to a kind of SDS precipitattion (carry over of potassium etc from media/PBS etc?), so centrifuged the sample > 10,000 x g for 10 min etc. Unexpectedly, instead of finding some precipitation at the bottom of the tube, there was some white marerial floating over the solution. The white material appeared to be some lipid but I could not be sure and do not know what it was and why it appeared (I never have observed such floating material after hundreds of cell lysates I made before). Any suggestions, please? Thank you.
Schu

#2 Trof

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Posted 03 September 2012 - 10:34 AM

What cell line?

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#3 bob1

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Posted 03 September 2012 - 12:44 PM

Could be DNA and protein that has precipitated out - wouldn't normally stay floating though.

#4 scddfmsku

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Posted 04 September 2012 - 03:07 AM

Thank you for your prompt replies.

> Trof
The cell line is human pluripotent cells cultured on Matrigel. The cells were trypsin dissociated, added with FBS containing culture media, centriguged,washed with PBS, centrifuged, then the pellet was snap frozen.
> bob1
Yes, the material floats. Do lipids also precipitate out?

Yesterday, I added urea to the cloudy cell lysate, then the solutoin became very clear and translucent. So it was something insoluble in the presence of SDS (4%) but was solubilized with urea (3-4 M). I believe the sample can now be quantified and analyzed by MS etc (although I still do not know what the material was). Thank you for your kind help and comments.

schu

#5 proteaMatt

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Posted 04 September 2012 - 12:28 PM

You will probably need to do some sort of clean up step as you prepare that sample for MS analysis, urea tends to inhibit ionization, although for that matter so does SDS.
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#6 scddfmsku

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Posted 04 September 2012 - 08:51 PM

> proteaMatt
Thank you for your comment. Yes, I will use the FASP (filter aided ...) method to prepare Lys-C/trypsin digested peptides to remove SDS (and others) then purify them with C18 to remove urea (and others). Thanks!
schu

#7 ascacioc

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Posted 06 September 2012 - 02:20 PM

I was thinking in the same lines as bob1: DNA? but it wouldn't float. Sometimes, in some cell lines (e.g. insect cells) you have loads of DNA in the cleared (centrifuged) cell lysate and you have to remove it by streptomycin sulphate precipitation. I never seen it float though.

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#8 scddfmsku

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Posted 11 September 2012 - 09:57 PM

> Andreea
Thank you for your comment. Yes, the material floats. I wodered if the density of the solution/lysate was higher than usual, but was not sure (used the same number of cells as I always had done). I did not know the "streptomycin sulphate precipitation" method. Thank you for the nice information!
Schu




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