Hi all.
I'm doing peptide (pI 9.1, no Cystein residue) expression from E. Coli.
And I purified it using IMAC with 300 mM imidazole/50 mM Na-phosphate/300 mM NaCl (the imidazole was injected with gradient flow).
I got a single main SDS-PAGE near the expected MW size (heat and non-heat).
But I have two main problems.
1. When I dialyzed it with Na-phosphate/300 mM NaCl (pH 8.0), I found a white colored precipitation.
2. So, I filtrate and buffer exchage it with PBS using Amicon centrifuge filter (3K). transfer the sup. to fresh tube.
I injected it into SEC (Size exclusion chromatography, SEC-5 Agilent) with PBS running buffer, pH 7.4), It was eluted earlier than antibody (~150 kDa) and another later imidazole peak.
When I injected the IMAC eluate into SEC without dialysis, I got a peak before antibody, again.
So I think my peptide was aggregated from cell lysis. But I don't know why I can get the single SDS band whithout heating.
To get a peptide without any aggrigarion, shoule I have to change the concentration of NaCl or add some detergent (triton X-100 or Tween 20) or change the pH?
I cannot sure what should I have to do more.
Examining the amino acid composition of the peptide could help you decide what approach to take to avoid its aggregation. For a highly hydrophobic peptide, you could include a nonionic detergent and avoid salt. If the peptide has many charged side chains, changing the pH by several units in either direction and increasing the salt concentration might help. Avoid concentrating the peptide any more than is necessary.
What is the size of your peptide? In your SDS wells do you see any aggregation traces? (normally you can see a blue intense band stuck between resolution and stacking gel). Otherwise, if you see a single band in the expected size, it may indicate the presence of oligomers in solution rather than amorphous aggregates (oligomers can be separated due to SDS, hence the band you see in your gel), maybe adding a little bit of Triton X100 in your buffer can be a solution
What is the pH of your buffer? You may try another buffer (like HEPES buffer pH = 8, since Na phosphate buffer works well around pH = 7.
Sometimes imidazole can cause your peptide to precipitate, you may want to be sure that what you see is not due to that.
Adam B Shapiro This is my peptide composition. I have many Gly, so the composition (percentage) is neutral ~41%, hydrophobic is ~30%, basic is ~20%.
The buffer's pH is 8.0 and the pI is 9.1. How can I add 0.1% of TritonX-100 or Tween 20 in lysis and binding buffer for IMAC?
Thank you :D
Jorgaq Pata The peptide is 10.8 kDa. I'll attach the SDS-PAGE image soon, I'm not in the lab not. There is no traced band only single band between 14 kDa - 6 kDa standard marker. Both heating and non-heating samples. I'll try to add 0.1% of Triton X-100 at the 50 mM sodium phosphate/300 mM NaCl for lysis and binding buffer for IMAC.
Should I have to add Triton X-100 for dialysis buffer after 300 mM imidazole/50 mM sodium phosphate/300 mM NaCl to remove imidazole?
Thanks :D
This is a small protein, not a peptide. Overall, it is basic. At pH 8, it probably has a net positive charge. Lowering the pH further, let's say to pH 6, might improve its solubility by partially protonating the His residues. Adding the nonionic detergent is worth a try, but consider the downstream effect of having it there. When you try to concentrate the protein, the detergent will also be concentrated. Consider using CHAPS detergent instead of Triton X-100. Because the micelles of CHAPS are very small, it will pass through the concentrator instead of accumulating. These detergents should not interfere with IMAC, and may actually improve lysis if their concentrations are high enough. The detergent concentration should be several-fold higher than the CMC (critical micellar concentration) during lysis. Afterwards, keep the detergent concentration at least a little bit above the CMC.
Adam B Shapiro Thank you :D I'll try at pH 6~7 with the same buffer composition. But the CHAPS, we don't have it and could not find it near other labs. When we get it, I'll try. Can I ask one more question?
I purify the peptide, eluted from IMAC and affinity chromatography. The eluate from IMAC, the SEC peaks is earlier than Ab, even though the sample buffer is different. When I tried to change buffer to PBS, there is a small imidazole peak.
But when I purify it by affinity chromatography and elute it by 100 mM Gly-HCl (pH 2.7) and neutralize it by 1M Tri-HCl (9.0), I got several peaks like the Figure.
The two eluates showed different SEC peaks. I want to ask your thinking about the results.
Thanks :D
Soi Yun Your SDS PAGE looks fine, for the dialysis lane, i cannot really distinguish the problem in your gel. However your SEC chromatogram is not so OK, since your protein elutes so quick. As for the affinity chromatography and the SEC, it is kinda normal that you don't have the same profile compared to the IMAC eluate, maybe you can do an SDS PAGE using the fractions of the other peaks to see if you can identify what it is or not.
Do you have any activity assay for your protein?
Jorgaq Pata I didn't think to get the fraction and do SDS-PAGE, I'll try.
I just think change pH or add EDTA (or my friends recommend for me how about add SDS).
I added a detergent (0.1% tritonX100) but, It was not effective for SEC.
There are many things I have to do more...
Actually, my thesis defense presentation is coming, next month.
Unfortunately, my protein has no possible activity assay, now. But I did SPR (binding affinity analysis to target) in HBS-EP (HEPES, EDTA, surfactant P20 included).
I couldn't check SEC at the time when I did SPR.
But the data showed it is bind to the target protein.
Thanks :D
It looks to me like you may be getting the peptide off the IMAC column as an aggregated form (12-13 min), and in a monomer or dimer form (~22 min). In contrast, the affinity chromatography looks like you got a wide range of aggregation levels.
Adam B Shapiro Oh I solved the problem :D. When I change the lysis and mobile phase buffer from 50 mM Sodium phosphate / 300 mM NaCl, pH 8.0 to 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, It has no aggregation.
And I single peak in SEC, the retention time was similar with the calculated time.
Thank you for your kindness :D
Actually, I'll have a PhD defense presentation in June. And It was my last data. Thank you again :D
Have a nice day!
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