Biotinylated peptide

Hello. I need to perform KD analysis on a biotinylated - peptide / mAB. I was planning on using a GE streptavidin chip to immobilize the biotinylated-peptide on the surface and pass on the mAB. The Mol. wt. of the biotin-peptide (1.5 kDa) is quite small and have not worked with molecules this size before. Do you have any recommendations on how much RU's I need to achieve in order to obtain good kinetics once I pass over the mAB (150 kDa) over it? I was also given another sample, a biotinylated protein with mol. weight of ~18 kDa. What would be the ideal immobilization level for this 2nd sample. Also, any suggestions on what to use for Regen solution? Thanks for your help!

Hi,

You can calculate the theoretically ligand immobilization response that is needed to have 200 RU of response when the analyte is injected with the formula at www.sprpages.nl/immobilization/immobilization-strategy

In your case this will be very low (3 RU) because of the big size difference between ligand and analyte. I would immobilize a little more (~25 RU to start with). When you have a nice response signal but the kinetics are mass-transfer limited you can try to immobilize a litlle less. I would recommend using the CAP immibilization kit since this kit is special developed for this kind of immobilizations.

In the case of a streptavidin chip it is not possible to remove the biotinylated ligand. For the ligand analyte interaction you should determine the regeneration conditions yourself.

The CAP-kit is designed to regenerate the whole chip including the biotinylated ligand.

Kind regards
Arnoud