Hi, i expressed a small peptide (~10 kD) with C-term His tag.
The target peptide was expressed with low solubility. So I make it solubilized with 2 mM EDTA.
I checked overexpressed SDS-PAGE band and checked His tag WB band.
After that, I purified it with IMAC resin and get purified peptide (I checked single band with SDS-PAGE).
After dialysis of the eluate, I did WB with Anti-His Ab.
However, I can't get any WB band.
I did 2 more times WB, there was no WB band.
Only after purification and dialysis, I can't get WB band.
Is there anyone who got same problem?
I can't sure, what should I do.
Thank you.
dear soi
give me some more details like
whats the concentration of Anti His ab used
did you check your protein in SDS page after dialysis
and brief about your WB protocol
If possible share the SDS PAGE before and after dialysis
EDTA can interfere with IMAC purification by stripping the metal ions of the column. EDTA is also not normally used for solubilising proteins, detergents or caotropes (e.g., urea) are better for this and compatible with IMAC.
Engelbert Buxbaum Thank you for your reply. Before I purify the peptide with resin, I removed the EDAT with dialysis (PBS) for 2 times. When I discussed with my advise professor about how to solubilize the peptide, I tried to use Urea or Guanidine-HCl. But, my professor didn't want to use Urea or Guanidine-HCl (I don't know the reason). He just recommended to me only EDAT.
Phani Kashyap S
Dear Phani kashyap.
Hi, thank you for your reply.
I check the SDS-PAGE and WB band after expression and dialysis.
For WB, I used 6xhis tag mAb Goat Anti mouse Ab-HRP from Thermo fisher and ECL substrate.
For purification, I used Qiagen superflow (NI-NTA) resin and pierce centrifuge column.
The supernatant (from sonicated e.coli cell) was dialyzed with PBS and mixed with imidazole/PBS (final conc. is 10 mM imidazole/PBS).
The 3 ml of mixed sup. was incubated with 1 ml resin for 2 h at 4 degree C and loaded to column.
(I keep the flow through)
The resin was washed with 4ml of 50 mM, 80 mM (2 times for each buffer, total 4 time) and elute with 2 ml of elution buffer (100, 300, 500 mM).
I checked the SDS-PAG target peptide band after elution.
And then, the eluate was dialyzes with 0.5xPBS.
The dialyzed eluate was loaded for SDS-PAGE and WB.
I got SDS-PAGE band, but no WB band.
I got WB band of dialyzed sample before purification and flow through, but only dialyzed eluate sampled had no WB band.
Thank you :)
Hi Soi yun
I think may be protein degraded in dialysis step and losses its His tag.
Why cant you try with specific antibody against you protein.
Phani Kashyap S
Hi Phani kashyap S.
My target peptide has no specific Ab, because it was screened from phage display.
I got WB from eluate (before dialysis) but I couldn't get WB band after dialysis.
I don't know what happened during dialysis.
I'll try WB again after dialysis with fresh sample.
If i have same problem, I'll make new question again.
Thank you:)
Have a nice day.
I hope that I can still be of use here, but as Engelbert has already mentioned: EDTA must have been the problem here.
His Tag Purification products like Ni-NTA are VERY sensetive to EDTA. Because EDTA, as a chelator, binds the metal ions like Ni that are used for the interaction with the HIS tag.
Because of that you are basiclly trying to purify with NTA instead of Ni-NTA.
As far as I know there is only one product out there that might help you here:
https://cube-biotech.com/products/purification-resins/his-affinity-resins/100-indigo-ni-agarose/
Its INDIGO-Ni, not Ni-NTA. The other Ligand binds the Nickel-Ion much stronger, enabling the use of EDTA concentrations up to 20mM (10x of the concentration that you have mentioned)
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