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  2. → Viewing Profile: Topics: qpwoei4756

qpwoei4756

Member Since 26 Dec 2011
Offline Last Active Feb 27 2013 04:26 PM
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Topics I've Started

EMSA: cell extract vs purified protein

08 February 2013 - 04:16 PM

Hi everyone,
I am planning to perform EMSA with a supposedly DNA binding protein and a 22-mer oligo DNA. But I was wondering what's the difference between using purified protein fraction versus cell extract extracts + noncompetitve DNA such as dIdC?
My protein is transiently transfected to human cells using lipofectamine 2000. I can already have my protein of interest purified from 293T human cells using anti-FLAG beads, but I can only get this much (see attached picture) protein. It's also time consuming and costly. I already tried to use the purified protein to do EMSA several times and I get little to no binding.
I'm reluctant to use the cell lysate because the expression may not be enough. Is this much protein expression enough for whole cell EMSA?

Thanks
qp

Protein Extraction: Extremely Sticky e coli lysate

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.

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