Posted 10 March 2010 - 09:17 PM
I'm working on a 30 KDa His tagged protein that purifies well on Ni-NTA and comes up in the soluble fraction. The protein however shows compromised activity and for long we hammered our heads against changing the assay conditions etc until we stumbled upon some gelfiltration and DLS results which show that almost 90% of the protein exists in the form of 'Soluble aggregates'.Can someone tell me a good method of breaking these aggregates??? High salt conditions just won't do because high ionic strength is known to hamper the activity.The buffer I use from the time of cell lysis contains: 10mM tris-Hcl(pH 8),10mM MgSo4,10mM Bme,5% glycerol,0.1mM PMSF. I have got a few random suggestions about going up in glycerol% upto 50 in the storage buffer and incorporating the substrate of this protein in the purification buffer to a conc of 1mM. How well would one recommend these suggestions??
Any suggestions or hints that would dissolve these aggregates would be highly appreciated as they might just bail me out of the most painful stage in my Ph.D
Posted 12 March 2010 - 08:24 AM
peg-8000 will co-concentrate on small pore membrane based concentrators (eg amicon ym-10) so we start low and let it increase to the desired concentration.
other things that may help are detergents (non-ionic such as triton, nonidet, etc.; ionic such as deoxycholate; not sds).
maybe ethylene glycol or dmso.
genius does what it must
i do what i get paid to do
Posted 16 March 2010 - 11:38 PM
Did you tried solubilization of aggregates with Chaotropic reagents( Urea, Gunidine HCl) cobined with Reducing agents (Beta mercapto ethanol). Use this combination with different pH. Normally, low amount of reducing agent should be used and pH would be higher values. This will be finalized based on results.
Have anice time.
Posted 24 March 2010 - 11:04 PM
all the tips you're getting are going in the right direction. It's difficult though to predict exactly what needs to be in the buffer to keep the protein from aggregating. The OptiSol protein solubility kit from Dilyx helps you finding your protein's solubility sweet spot. This is a way to systematically assess optimal pH, solubility enhancer etc.
You may want to give this a try.