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Help! Cell homogenization and protein extraction for Western Blotting


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#1 movement K

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Posted 07 February 2011 - 08:08 AM

I'm working with primary mouse hepatocytes.
Currently I'm having a trouble with protein extraction from my cell culture.

I usually add Tris-HCl lysis buffer (supplemented with sucrose, KCl, MgCl2) and sonicate the samples.
However, when I spin the samples in the centrifuge, no matter how fast I spin them down, I get white stuffs (guessing genomic DNA) coagulated on top of the cell lysate.
This gives me a so much trouble for Bradford assay and loading samples on gels for Western Blotting.

Is there any ways to get rid of them?

Thank you

#2 vivekp

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Posted 12 February 2011 - 11:26 AM

I'm working with primary mouse hepatocytes.
Currently I'm having a trouble with protein extraction from my cell culture.

I usually add Tris-HCl lysis buffer (supplemented with sucrose, KCl, MgCl2) and sonicate the samples.
However, when I spin the samples in the centrifuge, no matter how fast I spin them down, I get white stuffs (guessing genomic DNA) coagulated on top of the cell lysate.
This gives me a so much trouble for Bradford assay and loading samples on gels for Western Blotting.

Is there any ways to get rid of them?

Thank you


add some amount of Dnase....

#3 mdfenko

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Posted 14 February 2011 - 02:08 PM

i would think that the floating white material is lipid or carbohydrate.

Edited by mdfenko, 14 February 2011 - 02:11 PM.

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