Hi:)
I'm doing His tagged peptide expression and purification from E.coli.
I expressed the peptide and checked it with WB (with Anti-His Ab).
For purification, I did IMAC and got an eluate.
But it still necessary additional purification, because there were another impurity peptide bands in the eluate (I checked with SDS-PAGE).
We tried to use affinity chromatography (because the peptide has affinity domain with the ligand of the resin) and prepare the sample.
This is how I prepared the sample:
The eluate sample condition: suspended in 150 mM imidazole/50 mM sodium phosphate/300 mM NaCl (pH 8.0)
1. to remove the imidazole, the eluate was dialyzed with 50 mM sodium phosphate/300 mM NaCl for 2 times at 4℃ (first time: 6 h and second time: O/N)
2. the dialyzed sample was filtered with a syringe filter
: the filter I used was CA (cellulose acetate, PES, Nylon, and PVDF).
3. checked the concentration with nanodrop and loaded to SDS-PAGE
→ Here is the problem.
The concentration of the sample was decreased (more than half) and only the target peptide band was removed in SDS-PAGE.
And in the case of the PVDF filter, there was a low concentration of peptide band was detected (SDS-PAGE), others got no target band.
I sucked up the fluid from the filter and checked the SDS-PAGE band.
(please see the attached Fig.)
Additionally, I tried to use Amicon centrifugal filter (3K, 30K) and I lost my target protein again.
I think there are two possibilities.
A. the target peptide was denatured during the dialysis.
B. the target peptide adsorbed to filter membrane.
But... I don't know what I have to do to solve the problem.
Is there anyone who ever had the same problem as me? :D?
Thank you
Try using a filter with polyethersulfone (PES) membrane.
This filter material is widely use for the filtration of protein solutions.
PES is not listed in your gel photo.
What kind of second step are you planning to do?
If you cannot filter your sample, try high-speed centrifugation to remove any particulates.
Soi Yun PES looks a lot better than all the others.
Are you sure it removes a lot of your peptide?
You will not be able to find a membrane that does not bind some of your peptide.
Good day! Membranes adsorb a lot, see here:
Article Depot effect of bioactive components in experimental membran...
Look:
1. You need to determine peptide concentration at each step.
2. It may not be the filter problem!!
3. Either you have to lyophilize the sample, or you can do reverse dialysis to concentrate the sample .
4. If you still don't find the bands then you need to look at the buffers.
I hope you get the results.
Dear Achim Recktenwald Anju Kaushal
Thank you for your reply.
I summarized my results again and attached them by Figure.
We think the most problem is fragmentation and another is non-specific adsorption to the filter membrane (both PVEDF and Cellulose acetate type).
After purification and filtration or dialysis, peptide cleavage occurred and it was detected in WB.
And after that, the target peptide was removed (adsorbed to filter membrane) during filtration procedure, too.
I didn't try to lyophilization or reverse dialysis, yet.
And I think...
1. How about using a Protease Inhibitor Cocktail before purification?
2. How about change the suspension buffer (for e.coli cell suspension and purification buffer) from 50 mM sodium phosphate/300 mM NaCl (pH 8.0) to 20mM Tris-HCl, pH7.5 or 20 mM sodium phosphate or PBS ? would you recommend it for me?
(But, I think if there is no salt in the buffer, then the peptide will be aggregated. Because it has a hydrophobic part in C-ter.)
Thank you :D
I hope to take care of your safe from the Corona.
Anju Kaushal
Hi :D Actually, I tried to concentrate my sample with other buffers (HBS-EP; 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20. or 50 mM Tris-HCl, 150 mM NaCl, pH 7.4). But it lost my target protein again X).
And I dried 10 ml of my sample (in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4) and resuspend with 1-2 ml of Tris buffer. And then, dialyze it to remove high concentrated Tris- and NaCl. I checked its absorbance at 280 nm, It was minus (~-0.13). I got panic.
So, Now. I'm thinking decrease pH from 7.4 to 7 for protonation of C-term His of my protein. I cannot sure anything.
Soi Yun
Hello Soi,
You should not lose your target protein actually.
Certainly , there is a technical problem in your procedures I can pick this up as:
1. Check the protein conc. you are using in purification and also check how much protein you are losing in this process.
2. There is process to use filters , it seems you have your protein of interest is left on the supernatent.
Please work on these two factors carefully, you will soon find the results.
All the best
Anju Kaushal
I'm sorry to late because I got the thesis defense in theses days.
Actually, I changed the buffer to 50mM Tris-HCl, 150 mM NaCl, pH 7.4.
I checked the concentration after purification, it was around ~2.3 uM per 1L culture (0.04 mg/ml) with un-purified peptide.
And after filtration, the concentration decreased more than half.
In both phosphate and Tris buffer, it was disappeared during filtration.
The filtration step was performed with Amicon centrifuge filter for 3000 rpm, for 40 min at RT or 13000 rpm for 30 min. But all of the peptides was disappeared.
I didn't check the supernatant from the filter, yet.
I'll try it.
Thank you :D
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