December 14, 2019, 10:50:30 AM
Forum Rules: Read This Before Posting


Topic: how to resuspend E. coli bacterial cells gently  (Read 2613 times)

0 Members and 1 Guest are viewing this topic.

Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 3910
  • Mole Snacks: +244/-16
how to resuspend E. coli bacterial cells gently
« on: September 19, 2014, 09:45:10 AM »
We overexpress our proteins from E. coli cells then purify.  After centrifuging the cells from growth medium, we sometimes have trouble resuspending the cells in sonication buffer.  Long ago, I was trained to do this gently, by swirling small portions of buffer in the centrifuge tubes, but sometimes this takes an inordinately long time.  Yesterday we tried using a small paint brush and a gentle stroking action to resuspend them.  Does anyone have any suggestions for how to do this step without damaging the cells?

Offline Yggdrasil

  • Retired Staff
  • Sr. Member
  • *
  • Posts: 3207
  • Mole Snacks: +482/-21
  • Gender: Male
  • Physical Biochemist
Re: how to resuspend E. coli bacterial cells gently
« Reply #1 on: September 19, 2014, 10:48:34 AM »
I usually take a serological pipette and pipette up and down to resuspend the cells (squirting buffer at the pellet to disperse them and drawing larger chunks of cells to break them up).  This works well for E. coli, though it can also be a bit time consuming.

Offline Irlanur

  • Chemist
  • Full Member
  • *
  • Posts: 422
  • Mole Snacks: +32/-4
Re: how to resuspend E. coli bacterial cells gently
« Reply #2 on: September 19, 2014, 06:16:17 PM »
It's funny that you write this now. I'm not a biologist but I am learning the techniques now and this way of doing it is just absolutely annoying... people seem to do it that way tough. can't you just stirr it till it's off?

Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 3910
  • Mole Snacks: +244/-16
Re: how to resuspend E. coli bacterial cells gently
« Reply #3 on: September 20, 2014, 11:10:41 AM »
I am not sure what you mean by stirring till it is off.  When I first learned how to prep protein from E. coli, I was taught to do it by gently swirling a small portion of a small portion of sonnication buffer around the tube and slowly resuspending layers of cells in this manner.  IIRC the idea was that you don't want to break the cells prematurely.  It did take some time, however.  This week we followed a prep that called for a faster spin (8000 vs. 5000 RPM in a JA-10 rotor).  I think that the faster spin may have packed them down more tightly.  My idea of gently sweeping with a small paint brush was based upon something I had heard about post-sonnication pellets many years ago.  I don't have a good way to judge how gentle a technique it actually is.

Offline reeta12

  • Very New Member
  • *
  • Posts: 1
  • Mole Snacks: +0/-0
Re: how to resuspend E. coli bacterial cells gently
« Reply #4 on: September 29, 2014, 12:56:24 AM »
I'm gonna say yes, since the amino exchanger is polar charged. However there is also the ethyl groups? I'm not sure this is confusing me. How is my reasoning for the first problem if you don't mind me asking also

Sponsored Links