Hi everyone.. With my previous discussion I would like to add this point I'm trying to express and purify a 6X his tagged protein, I am getting a band on SDS PAGE at the desired mol wt size but I did not have the anti-his antibody to confirm the expression. Also, the protein is getting expressed as inclusion body and is almost pure and of the desired size and I use 8M urea for solubilizing it. But when I am doing Ni NTA purification my protein does not bind to the Ni-NTA column.The buffer also has 1mM B-Me.What could be going wrong? Please help. Thanks in advance.
Hi!! Shreyasi,
As per my experience in field of protein purification specifically for 6X His Tag i can suggest you to do following:
(1). First Sequence ligation junctions to ensure that the reading frame is correct.
(2). Might be your 6X His Tag has been degraded so you can re-clone and check again for expression and purification.
(3). Check pH and composition of all buffers and solutions. Dissociation of urea often causes a shift in pH. The pH values should be checked immediately prior to use. Ensure that there are no chelating or reducing agents present in your wash buffer and equilibration buffer.
(4). Use pH=8 for purification process. You can increase the pH slightly. But make sure about your protein pI as well. Your protein pI and pH should not be similar.
(5). Reduce wash stringency after passing your supernatent (crude protein lysate). Wash with less amount suppose 5-6 ml of 20 mM , 40 mM imidazole then elute your protein in 200-300 mM imidazole.
(6). After sonication you can concentrate your protein (make it around 1-2 ml ) then dialized your crude protein. then next day proceed it for purification.
(7). You can replace NI-NTA and try purification with fresh Ni-NTA.
(8). As you mentioned you don't have anti-His antibody which is necessary for confirmation of your recombinant protein. Better you purchase it and be more confident about your expressed protein.
(9). If all these suggestions won't work then go for ion exchange chromatography which is the best solution for these types of protein, but make sure about your protein. Sometimes there are some host proteins which may overexpress at your target protein molecular weight..
(10). Last thing you can try is that perform a second capture reaction means bind your protein with Ni-NTA resin (pass crude protein to column) then elute directly with 300 mM imidazole then concentrate it to 1-2 ml and dialised it overnight with specific pH and conc of buffer (which you use routinely). next day perform second step purification in Ni-NTA resin.
Hope these works for you. Give me you response if this would be helpful.
Good Luck.
Hi Gaurav
First thing is that we have not cloned the gene.We received the plasmid from a different lab. Second,is it possible that only the His tag is getting degraded?I have tried with buffers with 7.5 and 8 and with different salt concentrations also.I wash with 20mM imidazole and elute with 250 mM imidazole. But nothing is binding.Also every time I use fresh beads.Do these beads have any exp date and do they go bad?I check the pH everytime and maintain it between 7.5 to 8 and my buffer has 1mM B-Me.1 mM BMe should not reduce the resins I think. Plz help
Hi there,
If the protein is recovered in the flow through, it obviously shows no affinity for NiNTA so tag might have been degraded during expression/purification process. Check the proposal by WB using anti-His antibody.
Is it possible that only His tag is getting degraded??n the protein sequence is intact?
your resin would go brown if the resin had been reduced but you shouldn't really need a reducing agent in the buffer if you are using 8 urea.
try to get the protein expressed in a soluble fraction too if you can. What are your expression conditions?
in above to above points mentioned, I would suggest it looks like your protein is not eluting, try eluting with 0.5 and 2 M NaOH . do you know if the protein is N , C or Internal tagged? and for some proteins the HIS gets masked. or the protein is getting degraded fast due to proteases, so use protease inhibitors
Even though you solubilized your protein from inclusion bodies using 8M urea, it may still be in an aggregated state, and aggregated proteins often do not bind well to chromatography resins. Try running your protein on a gel filtration column in 8M urea and see if it elutes at the expected monomer molecular weight, or a much higher molecular weight. If the latter, the protein is aggregated. It may require stronger denaturing conditions to disaggregate it, such as 8M guanidine HCl (Also, make sure you use fresh urea solution, since urea is unstable).
Better still, as someone else suggested, try to find expression conditions that give soluble protein. Lowering the temperature to 16oC during expression is a good place to start.
Hi again,
Adam, I don't think during gel filtration run with 8M urea the denatured monomer will exhibit a mobility comparible with the one of the native protein obtained in native condition as the calibration is done with and valid for globular proteins...
Hi Dominique, I agree with you, but it should still be possible to distinguish an aggregate from an unfolded monomer, even though the monomer will elute larger than its molecular weight if it were globular. Also, you could avoid this problem by using unfolded proteins for the standards by using the same denaturant.
It is probably the Bme that is causing part of the problem. I have known my my proteins to flow through with 1mM. Try using TCEP or perhaps adding bme to your flow through fractions.
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