Hi,
I have expressed a protein in mammalian cells. It is a HIstidine tagged protein. I have tried to purifiy this using a buffer with imidazol in IMAC. This protein is very hydrophobic and adsorb bits of membrane, lipids, pyrogens, etc. making chromatography somewhat tricky. I have read by exclusion molecular and using a gradient up to 50% ethylene glycol in 0.1 M Citrate, pH5.0 the protein could be purified.
- Could I use ethylene glycol in my buffer with imidazol?
- Is it dangerous to work with ethylene glycol?
- Could glycerol work as EG?
- Could you give me any advice?
Thanks for your time!
Best wishes,
Kika
Hi Enriqueta casal-banciella . Ethylene glycol is safe to work with if it is not ingested and shouldn't interfere with IMAC. It will make the buffer more viscous and slow the column run.
Glycerol and ethylene glycol have similar properties when it comes to protein purification, but glycerol will make your solution even more viscous.
You won't be able to purify with citrate pH 5.0, chelation complexes work efficiently at pH 6-8.
What buffers were you initially trying, what were your salt levels?
Thanks a lot for your answers.
I would use citrate pH 5.0 if I would use size exclusion chromatography, but for IMAC I would use imidazol elution buffer pH 7.4
Kika
You need to elaborate on the buffers and the salt concentrations that you have been using already in your IMAC.
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