SDS PAGE of LB medium

Jayakumar R jakku71 at yahoo.com
Fri Feb 22 16:31:39 EST 2002


Mike..
You have to first spin the culture down and remove the cells. You can also
make it cell-free by using a filteration unit fitted with a 0.2 um filter.
Try using small volumes like 2ml or 10 ml cultures if your protein can be
present in sufficient volumes. The LB components can interefere in the run
very badly.  YOu may have to either concentrate the culture supernatant by
lyophilizing or by speed vacuuming.  Then dialyse the supernatant using a
dialysis membrane with a cutoff smaller than your protein of interest, so
that all the salts etc. get cleared off.  Then try to load as much of the
concentrate as you can, if you are not sure of the protein concentratin.
Protein determination by assays may not be of much use since the extracts
that you use in LB may give false results.  Other than TCA and acetone, I
read somewhere that 2 volumes of ethanol can precipitate proteins quite
efficiently in their native states.  Try that.
   best of luck
jayakumar

> From: "Emir Khatipov" <khatipovNO at NOuchicago.edu>
> Organization: The University of Chicago
> Date: Fri, 22 Feb 2002 14:55:23 -0600
> To: methods at hgmp.mrc.ac.uk
> Subject: Re: SDS PAGE of LB medium
> 
> I think TCA should work fine. As far as I remember, usually the amount of
> proteins bacteria excrete is in the milligram per liter range. You may also
> try precipitating proteins with acetone (usually <50% v/v); sometimes there
> is even no need to do it in the cold.
> Emir
> 
> "Michael Witty" <mw132 at mole.bio.cam.ac.uk> wrote in message
> news:Pine.SGI.4.33.0202181823310.1103697-100000 at mole.bio.cam.ac.uk...
>> Dear All,
>> I am going to try some SDS-PAGE of the medium my E. coli are
>> growing in tomorrow - to see if my desired protein is secreted into the
>> medium.  BUT how much should I load?  I am going to TCA ppt some to avoid
>> salt, but should I expect a large amount of spurious protein or hardly any
>> at all?  Plan A is to load ppt from 25microlitres per gel lane.
>> Regards, Mike.
>> 
> 


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