Hello everybody
My peptide has MW of 5 kDa, to be conjugated with PEG-SH 10 kDa via SH-maleimide interaction at high pH. This is not a specific binding, so, after fisnishing reaction, I have 3 isomers in my sample (mono PEGylated-peptide, peptide-peptide and PEG-PEG), there would be 3 kinds of different molecular weight in my sample. Can I use ion exchange chromatography to separate 2 isomer before apply to size exclusion chromatography?
My peptide pI is 4.2, the reaction pH is 7.0. For purification, I used acid citric pH 3.0 for cation exchange chromatography and Tris-HCl buffer pH 7.5 and 9.5 for anion exchange chromatography, CIM SO3 and CIM Q column are used. However there are too many peaks in the result and they are not separated.
Can you suggest me how I can purify my peptide to get only mono-pegylated peptide? Thanks in advance
Dear Mr Zhao,
Yes, I used desalting column before doing ion exchange chromatography.
Dear Diem Phuong,
If you want to get single peptide, I think you need use FPLC and HPLC. This method will separation peptide with some peaks, then you can select one peptide.
1. Separated peptide followed by MW using MW standard
2. Chose peptide with MW that you need ..(1-3kDa; 3-5kDa; 5-10kDa) peptide (I)
3. One more separation peptide (I) with some buffers, and now they only show some peaks.
Good luck ^^
Dear Diem
I think its better you first separate the reaction mixture in size exclusion chromatography based on size, and fractionate them and then try the fractions in IEX and compare the results with the result of reaction mixture in IEX to see which one might be mono PEGylated. By this way you can have an idea that which peak you need to purify.
For purification I think anion exchange might work better. You may need to optimize the condition based on the preliminary results you have got. If you let me to see the chromatogram, I may guess which parameters should be optimized.
Good luck
Dear Diem
One more suggestion I have for you. As you are separating peptide, why you are not trying the Hydrophobic interaction chromatography. Have a quick search in web and try it too if you have column.
Thank you guys, I've tried it several times again and again and finally succeeded. Thank you for your help~
Best,
Nguyen Diem
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