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Mammalian Cell Lysis


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#1 Mafer

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Posted 14 March 2017 - 11:34 PM

Hi,

 

I am trying to lyse cells (HEK and CHO) for protein (recombinantly expressed). I've ran an SDS-PAGE using cell samples in 2% SDS and clear band showing my protein being expressed (quite a lot), but when I try using some lysis buffer, and centrifuged them - supernatant was ran on gel and the band was not there.

 

So far, I tried using (1) M-PER, (2) 150mM NaCl, 50mM Tris with 1% detergent (NP-40, Triton X-100, Tween 20 and Tween 80) (3) 50mM sodium phosphate, 300mM NaCl, 10mM Imidazole with 1% detergent (NP-40, Triton X-100, Tween 20 and Tween 80)

 

None of the above worked. Wonder if there is any other way to lyse them effectively.

 

My final goal is to extract them in native form as I would like to isolate protein by His-Tag Isolation. I've run the lysed supernatant from above through His Tag isolation and can actually elute some of my protein. So my assumption is that my lysis procedure is not optimal. Is what I think logical?

 

Thanks.



#2 mdfenko

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Posted 16 March 2017 - 03:47 AM

the protein of interest may be in inclusion bodies.

 

your options:

 

you can try to perform the expression incubation at reduced temperatures (may work)

 

suspend the pellet in a medium containing urea to denature the protein, isolate with the his-tag then renature by slowly reducing the urea concentration (either on the column before elution or by step-wise dialysis against reduced urea after elution)


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