I am conjugating a small cysteine-containing peptide (~700 Da) to a 4-arm PEG-maleimide (20 kDa) using thiol-maleimide chemistry.
I will add an excess of peptide so that virtually all maleimide groups are reacted with.
What would be the best way to purify these conjugates and obtain 4-arm PEG with peptide conjugated to each of the four arms?
A post-doc in my group recommended dialysis with a 10 kDa membrane cutoff, however all papers I am finding online use chromatographic methods like HPLC, FPLC, SEC, cation-exchange chromatography, etc.
Is there a particular reason why no one uses dialysis? Does chromatographic purification just ensure a much higher yield of the final purified product?
Thanks in advance.
Hi Ashley,
I believe dialysis is a good method to perform a pre-purification to eliminate molecules with a lower molecular weight than the PEGylated protein and then, carry out a second purification step using a chromatographic method to separate molecules of similar molecular weight based on others physicochemical criteria.
Dear @Ashley,
in principle dialysis works for your project; I often use this technique for size separation. An alternative could be a concentrator.
But keep in mind, that PEG is somehow consisting of linear molecules, and maybe there is a deviation from "spherical state".
And the MWCO is mentioned for spherical substances.
So you should decide for a smaller dialysis membrane, e.g. with a MWCO of 1 kDa, otherwise you'll lose too much product. And keep in mind, that you have to change the buffer quite often (8-10x) in order to get a pure product.
If you want to dialyse, your best option is to use a 3KDa MWCO membrane, which is small enough to retain a 20kDa molecule (~23 kDa after conjugation) and large enough to let free peptide through. I'm suggesting 3 kDa rather than 1 kDa membrane because some of your peptide may be present as disulfide-bridged dimers at 1.4KDa size. A 5 kDa membrane would also work but usually the step in sizes with these membranes is 1, 3, & 10 kDa.
If your sample volume is 1/100 of the dialysis buffer volume, AND if you allow equilibrium to be reached with each change of buffer, just 4 changes of buffer will reduce the concentration of the peptide by a factor of 10^8.
An alternative to dialysis, esp. if your sample volume should be small, is gel filtration on Sephadex G-25. NAP-5, PD-10 or similar columns don't need any equipment and may be operated by gravity flow. .
Ultrafiltration (UF), dialysis, and chromatographic methods work...Physical separation by size can be completed using SEC resins and membranes incorporated with UF device (centrifugal or stirred cell) and dialysis bag. However, HIC is also effective to separate pegylated hydrophobic substances. But you should get rid of excess salt typically ammonium sulfate as the second step. In chromatographic methods both for SEC and HIC amount of the load is critical.
For this, if the volume is high and the amount of the isolate is critical, I would prefer UF or gel filtration (large volume desalting mode) as gravity flow format for obtaining rapid enrichment, clean up and buffer exchange. Keep in mind highly hydrophobic substances such as PEGylated proteins and peptides may retain non-specifically to most SEC resins and additional organic mobile phase compositions should be used. Therefore it is indispensable to choose a highly polar polymer with a proper exclusion limit.
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