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Protein Extraction: Extremely Sticky e coli lysate


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#1 qpwoei4756

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Posted 04 December 2012 - 04:09 PM

Hi,

I'm trying to extract a 6x his tagged protein from BL21 competent cells, but the pellets are so sticky I can't collect my protein.
What I did was induce my protein using IPTG for 4hr, pelleted them and placed it in -20C. So far everything's fine.
When I took it out three days later and thawed it, however, it looked sticky just by shaking the tubes.
They are sticky as snout and no matter how much I mix it with my lysis buffer, it doesn't dissolve.
I tried adding DNAse, sonicating for a longer time, vortexing, and centrifuging them at twice the normal speed, but the stickiness won't go away. I can't get the supernatant, where my protein would have been located.

Why is this happening? Should I try to collect my proteins without ever freezing the cell pellets? Thank You.

#2 PhDinAcronyms

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Posted 05 December 2012 - 03:54 PM

If the stickiness happened after lysis, I would think it's DNA. Did you also add MgCl2 with the DNase? Needs magnesium to work




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