Poor fit

Hello. I am getting a poor fit on my Strepavidin experiment. (Photo 1). Immobilized Anti-IgG Fc specific Biotinylated Ab on a SA (GE) chip. After immobilization. blocked other sites with amino-PEO-Biotin at 50uM. I then passed on the Analyte, a mAB, at the following conc. (0.16, 0.8, 4, 20, 100nM). I used the same diluent in preparing the samples (HBS P+ / 0.01% BSA). I also noticed an inverted response when I just run HBS over the surface (Photo 2). Finally, I did read somewhere to include a washing step 50% IPA in 1M NaCl and 50mM NaOH after each ligand injection, is this necessary? I did not do this and was not so sure if this is standard practice in using the SA sensor chip.. Any comments / suggestions are appreciated...
Thanks.

Hi,
The extra washing seems a little harsh to me. It could damage your ligand on the chip. The purpose is probably to get rid of electrostatic bound ligand. Some cleaning is probably good to do but use less harsh conditions (e.g. 1M NaCl in buffer pH ~8.5-9).

The fitting is not following the curves probably because the curves are not 1:1. Normally I 'built' the fitting by starting fitting the dissociation alone, then association + dissociation with no RI, then ass. + dis. + Ri. Use the obtained values from the previous fitting as starting values in the next (you can fix them first and then float during the fitting).

Arnoud