Hello everyone, I am doing a batch-purification of a His-tagged protein using the His Select Ni Affinity gel and seem to losing my protein during the washes. The protein is present in the supernatant after bacteria lysis and after the first wash with buffer containing 50mM Phosphate, 300mM NaCl and 10mM Imidazole there is no signal.
How do I correct this problem?
I agree with the comments from Charles and would like to add a couple more:
1) Before IMAC I would run a western blot to make sure that your His-tag is present on your protein and is accessible (sometimes the location of the tag makes it not accessible because of steric hindrance due to protein conformation).
2) If you do not use protease inhibitors during lysis step you tag might be proteolytically cleaved from your protein as a part of proteolytic degradation.
3) Try to use lower concentration of Sodium Chloride (from 300 mM to 150 mM).
4) As mentioned by Charles you need to make sure that the presence of your protein in the supernatant is not a result of your column saturation (limit of capacity). In another words, that your protein can not be eluted from the resin with 300-400 mM Imidazole.
5) Make sure that your lysis buffer does not have any EDTA (it will bind Ni on your column and prevent binding of your protein.
Hi Robert,
If I understood your problem well, you did not recover your protein during your elution step after the wash that you described.
A couple of questions then:
¤ Is it the first time that this His-tagged protein is being purified; in other words: are you sure that the His-tag is available and working?
¤ Did you look for your protein in the wash and in the flowthrough after your IMAC? If so, it should give you clues as to whether your wash is too strong (and everything elutes in it), and whether your column is properly activated (and everything stayed in the FT).
Good luck!
I agree with the comments from Charles and would like to add a couple more:
1) Before IMAC I would run a western blot to make sure that your His-tag is present on your protein and is accessible (sometimes the location of the tag makes it not accessible because of steric hindrance due to protein conformation).
2) If you do not use protease inhibitors during lysis step you tag might be proteolytically cleaved from your protein as a part of proteolytic degradation.
3) Try to use lower concentration of Sodium Chloride (from 300 mM to 150 mM).
4) As mentioned by Charles you need to make sure that the presence of your protein in the supernatant is not a result of your column saturation (limit of capacity). In another words, that your protein can not be eluted from the resin with 300-400 mM Imidazole.
5) Make sure that your lysis buffer does not have any EDTA (it will bind Ni on your column and prevent binding of your protein.
I agree with the comments of Viktor and Charles
Perhaps another thing to do is to verify that your protein is fully soluble:
- did you made a high speed centrifugation (i.e. 100 000g) to check that your protein is still in the supernatant ?
Try to use 30mM Phosphate, 100-150mM NaCl and 10mM Imidazole in your wash buffer. Check flowthrough fraction after IMAC (Western blot).
Hi Robert,
I agree with above comments, but another hing is charging your column. be sure that your column get charged( you can realize by column color, nearly blue), and pH of your wash buffer that you didn' mentioned,it has a great role in protein elution.
good luck
Thank you all for ALL the suggestions. I will follow them up and feed you back. Good luck to you too.
Just to add, the main problem is to see, where is your protein - in the wash or still on the resin?
You can also boil little of your resin and load that to SDS-PAGE and do blotting to see, whether it's still there.
And mainly, omit the imidazole.
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