hello,
I am currently using LDH protein and have so far made a mixture of the LDH solution (in a phosphate buffer) and toyopearl chromatograph particles. Now, I want to determine how much of the LDH is immobilized onto the chromatograph particles before I add any binding reagent. At first I taught about using UV-Vis and determining the concentration using a standard curve. However, I am worried about the effect of the toyopearl particle slurry in the solution.What is the best method for comparing the concentrations before and after? thank you!
Hi,
When immobilizing a protein or peptide, the coupling efficiency can be calculated by comparing the concentrations of the protein solution before to that of the solution after conjugation to the beads. In that sense, the decrease in concentration is a measure for the amount that was coupled to the beads. If you know how many beads (active positions) you started out with, you can determine the amount of coupled protein (per volume of beads).
Good luck!
Thank you for your response Mr. Dirksen. If I may ask some questions, so is it ok to use UV-vis to determine the concentration of the solution? And also can you elaborate on the idea of active position? Thank you!
Yes, you can use UV-Vis, as long as you make sure that all beads are out of your sample (so after briefly spinning the samples in a centrifuge).
What I meant by active positions is that the manufacturer of beads normally provide some information with respect to the amount of active groups that are on the beads, or the amount of protein/peptide that can be bound to 1 mL of beads. This information will eventually help you determine how much protein/peptide is bound to 1 mL of beads.
- Badges
- Science method