I have purified a protein of 36kDa with Ni-NTA affinity chrmotography and initially protein was eluted with 100-500mM imidazole.
When elution was started with 300mM imidazole and so on protein was eluted in precipitated form.
I have changed buffering conditions like 50mM Tris pH 8.5, phosphate (7.4)and 25mM HEPES(7.4) and 5% glycerol and 2mM DTT was included in the elution buffer separately but there is no change in the precipitation.
Even 500mM NaCl in the lysis and elution buffer have not shown any effect.
Protein eluted with 100 and 200mM imidazole is stable and low in quantity compared to 300-500mM elutions.
I needed to go up to 8mg/ml of protein for my further studies.
Provide your valuable suggestions to overcome the problem.
Lysis buffer : 50mM Tris,300mM NaCl,0.1 % Triton,1mM PMSF,10 % Glycerol.
Protein pI : 6.5
Perhaps you can improve the yield of soluble protein by changing expression conditions, i.e. growing at lower temperature, using different expression strains etc. Maybe the Ni-NTA step is not the problem, but the step before that. To check this you might assay the lysed cell extract somehow for the fraction of aggregated protein. For example by high-speed centrifugation.
That reminds me, we once had a protein that was cold-sensitive, in this case we had to do the purification at room temperature!
You didn't mentioned the temperature, you purified protein. Better is to carryout protein purification at 4 C, you can also consider using 100-200 mM imidazole for elution. Also, expression at lower temperature helps, I would say 16 C. Hope it will help.
I have purified my protein at 4C and even at room temperature and protein was expressed at 16 C,centrifuged my cell lysate at 12000rpm for 30min.
Have you tried to add more NaCl to your protein after elution? Maybe your protein needs higher than 500mM NaCl to stay soluble? I had a protein which was really unhappy in lower salt concentrations (around 150mM); and it precipitated whenever I was trying to concentrate it. So when I added NaCl, the protein got solubilized and the precipitate was completely gone!
Also I would suggest that you do a buffer screen for your protein to find out what buffer conditions are most suitable for your protein. Good luck!
a couple of options drop pH, use EDTA or L-Histidine or use a cobalt column such as Talon or just strip a NTA column with EDTA and charge with Cobalt. We find L-His works well, keep your washes with imidazole
Can i use other buffer components (50mM Tris,300mM NaCl) along with L-Histidine ?
yes just exchange the imidazole for 150-200mM L-His just make sure you pH the buffers after the addition of the L-His
Thank you James,
Elution with histidine reduced the extent of precipitation...
I have started my elution from 200-300mM Histidine,but fractions eluted with the 200mM Histidine have shown low amount of precipitation compared to 200mM imidazole (my protein is happy with Histidine).
But still I have to go trough the elution with 150mM Histidine for further optimization.
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