Insoluble GST fusion protein help

Michael O'Hare mohar052removethis at uottawa.ca
Thu Feb 19 14:43:11 EST 2004


Your help is very much appreciated Nina.  I will put your suggestions to 
use.  Thank you

Michael

Nina Baltes wrote:
> Michael O'Hare wrote...
> 
>>Hi.  I need help purifying a highly insoluble GST-fusion protein.  I 
>>don't neen lots of it, just enough to serve as substrate in a kinase 
>>assay.  When I test my extracts from IPTG-induced vs non-induced I don't 
>>see any differences in the banding pattern.   Any advice would be 
>>appreciated.
> 
> 
> If by insoluble you mean that the fusion protein forms aggregates in the 
> cells, here's a protocol that isolates these:
> 
> - Induce a fresh 50 ml culture at OD660 = 0,3 - 0,5 with 1 mM IPTG for 2 
> hours
> - Centrifuge bacteria at 5000 rpm for 10 min 
> - Resuspend cell pellet in 1-5 ml in 50 mM Tris buffer pH 8.0 with 25% 
> sucrose 
> - Freeze at -70°C for 30 min or over night 
> - Thaw, then add 0,25 ml lysozyme (10 mg/ml in 250 mM Tris buffer, pH 
> 8.0) put on ice for 10 min.
> - Add 10 ml 2x RIPA/TET (5:4, see below), put on ice for 5-10 min
> - sonicate: duty cycle 50%; timer 1 min, full power; until solution is 
> thin and opalescent; don't let it warm up!
> - Centrifuge for 20 min at 15.000 rpm 
> - resuspend pellet in 500 µl A. dest.
> 
> The pellet should be clear, sometimes there's also some strange 
> insoluble black material in it, but this could be lab-specific ;).
> 
> 2X RIPA 
> 
> 20 mM 		Tris, pH 7.4
> 300 mM 	NaCl
> 2% 		Na-Deoxycholic Salt
> 2% 		NP-40 (Tertigol)
> 
> TET 
> 
> 100 mM 	Tris, pH 8.0
> 50 mM 		EDTA, pH 8.0
> 2% 		Triton-X 100
> 
> 1 µl of the resuspended aggregate usually gives a fat band on an SDS 
> minigel, with little "background". However, it's quite insoluble (duh), 
> so you'd probably have to experiment with different resuspension buffers 
> or treatments to standardize the amount for your enzyme assay (I've 
> never tried this though, so I can't help you there ;)).
> 
> Good luck!
> 
> Nina




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