pH scouting - No complete dissociation.

Hi there.

Machine: Reichert SR7000dc
Chip: Xantec polycarboxylated hydrogel (HC) 200M.
Protein solution - 5 mM sodium acetate pH 4-5 + 25 µg/mL IgG antibody (pI 6.4-9.0 ?)
Running buffer - degassed DI water
Cleaning solution




I've seen from other pH scoutings that the measurement return immediately to baseline after the protein solution injection ends. In my case, this doesn't happend. I've read that this suggest too low pH and denaturation of protein. Is this the case in my experiment, or what do you think? And what would you do to solve it?

Also, upon 'pump refill', it returns to baseline in right channel (red) even before cleaning solution injection.

Best regards,
Layzing

Hi Layzing,
Because you use an antibody I would recommend to use a buffer instead of DI water. A buffer (Hepes, Tris) with 150 mM NaCl will lower electrostatic interactions with the matrix and maybe the baseline will return.
Do you know the content of the cleaning solution? In Biacore 50 mM NaOH is used to clean the chip before immobilization. It should be compatible with the Xantec HC chip.

Arnoud

Hi Arnoud.

Thank you for the reply.

During the pH scouting I swapped to PBS buffer as the running buffer, and as you imply the measurements now return to baseline after injection. You think I should use PBS running buffer under immobilizatio, despite Xantec immobilization protocol states that immobilization should be performed with dd water as running buffer?

Yes, the cleaning/elution buffer consist of 1 M sodium chloride in 0.1 M sodium borate pH 9.0, but it seems to work just fine as is.

I am currently working on my master about kinetics of GLP-1 and various GLP-1 antibodies interactions and epitope mapping. I am not the most experienced with SPR, so I appreciate all the help you can provide. Thanks for now, hopefully you'll be willing to assist me upon future complications.

Best regards,
Lasse