GST fusion proteins affinity experiments.

[Ian A. York]

JIn article <rmrrgc.11.000E8039 at leeds.ac.uk> rmrrgc at leeds.ac.uk (R.G. 
Chaudhari) writes:
>Does anyone use GST fusion proteins to analyse protein interactions in 
>precipitation experiments. I am incubating a GST- viral structural protein 
>attached to glutathione S sepharose beads with infected cell lysates, spinning 
>down the beads, washing them and resolving interacting proteins by SDS-PAGE. 
>Unfortunatly  the same proteins are being resolved when  a GST attached to the 
>beads control is used. Does anyone know the problem, can anyone help or better 
>still, can anyone send me a protocol that has been proven to work v.well. 

There's no simple way to do this, because every protein-protein 
interaction has different requirements.  Here are some variables to think 
about, if you haven't already done so:

-labelling conditions.  Make sure you get a wide variety of cellular 
proteins labeleed.  For example, try a long label (overnight) and then 
spike again for an hour to pick up short lived proteins.  Depending on 
the virus you're working with, this may not be a factor.

-cell lysis conditions.  This also affects binding conditions.  In 
general, you probably want to start with very non-stringent binding 
conditions and try to increase the stringency.  If possible, it's 
probably worth running several conditions together, to see if one of them 
allows you to see specific conditions over your background.  

The two things affecting stringency - at least the ones that are easiest 
to adjust - are your detergent and your salt concentration.  Some 
detergents to try in order of least to most stringent are -
digitonin
octylglucoside
NP-40
deoxycholate
SDS

and you can vary concentrations of these as well.  (There are many other 
detergents you can use, these are just some that I've had experience 
with.)  

As far as salt concentration, try [NaCl] at, say, 0, 75, 150, 250, 500 mM. 
150 mM is sort-of physiological.  Some interactions are enhanced at high
salt, but these are unusualy as I understand it.  If you are really
frustrated, consider making up a salt mix that corresponds to the ions in
cytoplasm (can't remember the mix right now, I think I got one from
Methods in Enzymology Protein Purification). 

To lyse your cells without detergent, you can use a number of means.  
Dounce homogenizer is probably the simplest, especially for small 
samples.  

Remember to add a good mix of protease inhibitors.  

Finally, GST-fusions simply may not work, if the conformation is too far 
off because of the fusion.  Similarly, a seemingly specific interaction 
may be meaningless.  You should have some backup methods of demonstrating 
relevance as well.

Good luck.

Ian
-- 
Ian York   (york at mbcrr.harvard.edu)
Dana-Farber Cancer Institute, 44 Binney St., Boston MA 02115
Phone (617)-632-3921     Fax  (617)-632-2627


-- 
Ian York   (york at mbcrr.harvard.edu)
Dana-Farber Cancer Institute, 44 Binney St., Boston MA 02115
Phone (617)-632-3921     Fax  (617)-632-2627