betaine/sorbitol questions

p.j. van santbrink santbrin at CHEM.LEIDENUNIV.NL
Tue Nov 12 11:10:08 EST 1996



Dear netters

I've read that by using betaine and sorbitol in
your medium you have a better change to get a 
fusion protein in the soluble fraction after lysis 
of your bacteria (and not in inclusion bodies).
I'am using the pGEX system of Pharmacia and in 
pilot experiments I indeed managed to get both
GST and GST+protein in the soluble fraction after 
lysis.
But when I tried to get other fusion proteins in 
the soluble fraction by the same method I didn't 
succeed (almost everything in the insoluble fraction).
Those other fusion proteins are very similar to the
one I did get in the soluble fraction so I'm really 
puzzled.
Could somebody tell me more about this betaine/sorbitol
method and tell me the essential steps in the procedure 
(f.i. how important is the OD600 at which you induce your bugs).

Protocol I use, short version (send E-mail for detailed 
protocol if you need it to give an answer to my problem):

- plate bugs from -80 stock (not thawed) on LB-agar plate
  suplemented with amp, 2,5 mM Betaine and 0,1 M Sorbitol
- pick free colony from plate and grow (overnight)in 
  LB + the above mentioned suplements.
- dilute O/N culture 1/10 in fresh medium and incubate
  1,5 hour 37 degrees Celsius.
- put bugs at room temperature and induce with IPTG (final
  concentration 0,5 mM): incubate at 30 degrees Celsius,
  225 rpm for 2 hours.
- lyse bugs with lysozyme (1 mg/ml) and 0,2% triton X-100
- after lysis centrifuge (14 k, 10 minutes): supernatant is
  soluble fraction, pellet is insoluble fraction).
- bring part of fractions on SDS PAGE gel to look at results.

I hope somebody can help me.

Peter van Santbrink
University of Leiden
The Netherlands
E-mail: santbrin at lacdr.LeidenUniv.nl


"Is a dream a lie if it don't come true
 or is it something worse ?"

Bruce Springsteen, The River 






More information about the Methods mailing list