Only a small amount of protein is comming in soluble fraction even at 18degree with 0.3mM IPTG. On purifiying with both Ni NTA and glutathion beads it is coming out in flow through and wash fraction and instad some other nonspesific protein are coming on elution. pH of buffer is 7.0 and i have tried with both PBS and TBS and I have also kept it for 45 min at 4 degree with affinity resin . Kindly suggest me to get them bind through the affinity Tag.
Hi Rani,
for the His-tagged protein my first suggestion is to increase the pH of the binding buffer to 7.5-8.0. This should help with the binding, although you need to be careful and watch for precipitation (the pH would be very close to the pI of your protein). Also, if you have any imidazole in the binding buffer (it's common to use 20-40 mM to reduce unspecific binding) you may want to remove it.
If the protein still doesn't bind, it possible that the N terminal His-tag is inaccessible for binding. The His-tag is small, so it happens sometimes that it is "covered" by the folding of the protein. So you could try to move it at the C-terminus.
I have no previous experience with GST tag, so I'm afraid I cannot be of much help with that. However, you can give a look to the handbook of the resin you are using, as it may have some good suggestions for troubleshooting. For example, the manual of the GSTtrap column from GE Healthcare suggests to add some DTT to improve the binding, or also to reduce sonication times to avoid denaturation of the tag.
If you still have no luck with affinity chromatography, you could try with a different approach. For example with ion exchange. Since the pI of your protein is 9.05, at pH 7.0 it should bind a cation exchange resin. You would probably get a few contaminants, but it's better than nothing and you can always add a second purification step!
To start with, 16 -18 degrees is good, but the concentration of IPTG is very low. Try 1mM IPTG.
Also you do not mention anything about the method of protein extraction used. this will be crucial even before purifying the His tagged protein.
Be mind that most cellular protein has protease enzymes from the host, which could break down your protein of interest. Try using Bugbuster + EDTA free protease inhibitor cocktail specific for His tagged proteins.
You could also use 10mM of imidazole in your extraction buffer (needs optimisation).
Most purification buffers, Tris-HCl, sodium phosphate based would work well at neutral pH pf 7.4 -7.5 with gradient elution.
Good luck
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