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I will use IEX chromatography to purify a protein and will then use a equilibration buffer Tris-HCl, pH 8 to set the start conditions for my column and optimize the binding of my protein. Then inject my sample and further on elute with the same buffer but with a salt gradient.

My sample will contain my protein as well as the PBS buffer (pH 7) that I used in previous purification steps.

This question might be dumb but is this applicable, i.e. can I inject my sample with the PBS buffer to the column after equilibration with another buffer, or do I have to change my sample buffer in some way before the chromatography?

Thanks!

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    $\begingroup$ Depends on the injection volume. If the sample volume is very small, then it should not matter. IEX peak shape is dependent on the sample solvent in some conditions. Try the experiment at a small scale. $\endgroup$
    – AChem
    Jun 29, 2019 at 13:56
  • $\begingroup$ OK, thanks! But is it standard to have the same buffer for the sample as well as the buffer for the chromatography? $\endgroup$
    – Michelangelo
    Jun 29, 2019 at 14:23
  • $\begingroup$ Are you doing preparative HPLC or analytical? What are the column dimensions? Is it a simple glass column IEX. What is your sample volume? $\endgroup$
    – AChem
    Jun 29, 2019 at 14:35
  • $\begingroup$ Bed size is 7 × 25 mm, volume 1 mL. Sample volume, I don't know. I suppose it is preparative since my goal is to obtain fractions of my specific protein. $\endgroup$
    – Michelangelo
    Jun 29, 2019 at 14:57
  • $\begingroup$ I would say stick to the correct protocol you column volume is very small. $\endgroup$
    – AChem
    Jun 29, 2019 at 15:07

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