Unusual sensorgram shape

Hello. I ran this following experiment. Immobilized an anti-flag Ab to a CM5 using NHS/EDC. RL at ~12000 RU. Then injected Ligand, has a Flag tag (Mw: 85.9kDa). RL at ~325RU. Then passed on Analyte (Mw: 35 kDa). using HBS P. at conc. of 100, 20, 4, 0.8, 0.16nM. I am seeing the dissociation go down, then would go up. U-shaped. The figures below shows different sensorgrams. (Figure 1): FC 4-3. All conc.; (Figure 2): FC 4-3: Ligand injection followed by a sample blank (HBS); (Figure 3): FC 3, reference blank with HBS injected; (Figure 4): FC3, red, reference , FC4, green, 100nM sample. Any suggestions on how I can address the drifting issue? Is this the reason for the unusual dissociation shape? Just FYI, I did a desorb and sanitize 3 days for this run and let it flow at standby for these 3 days... Thanks.

Drift works in two ways. Drift in the reference and drift in de active channels. From figure 3 it can be seen that there is drift in the reference. But when subtracting positive reference drift you expect the active channel to be more negative. Since this is not the case in figure one, the drift is possibly mainly due to the active channel. Since the active channel contains a lot of ligand, this can lead to some drift. Especially when you use some form of regeneration. When using regeneration on a high density surface a longer stabilisation time after the regeneration may be helpfull to wash out all the chemicals and stabilize the system again.
We standard add a stabilization flow of five minutes before starting the first injection. On a T200 you have to 'fake' this by injection of buffer and add a wait time since a 'pause/wait' command is not available.
A lower ligand density may also be better to lower drift. It seems that you have enough capacity/response to bind the Flag-tag protein to lower the anti-Flag-tag antibody.
Kind regards

Arnoud

Thanks Arnoud, I will try your suggestion.

Hi Arnoud, seems like the extra washing did not help. I will try the lower ligand density. In did think about something. The submitter informed me that the Analyte (35kDa) has a his-tag C term. How about reversing the reaction? Meaning immobilize the Analyte unto a chip and then pass on what used to be my Ligand (85.9 kDa). Would that be feasible?

You can try to capture with an anti His or NTA chip.
Reversing an interacting pair is always an option when encountering non-specific binding or drift by one of the interactants. Try to immobilize not too much ( www.sprpages.nl/immobilization/immobilization-strategy ).
Arnoud

Hi Arnoud, I was thinking of using the anti-his kit. But found out both the ligand and the analyte has a his tag. But thought about doing the following...Immobilize the anti-his on a CM5 chip using NHS/EDC. then pass on the Ligand. Then once bound, I was going to flow a reagent (a blocking reagent), to block all other open anti-His sites. Then, pass on the Analyte which will bind to the Ligand (hoping all the unoccupied anit-his sites are blocked, so that the interaction is only between the bound Ligand and the Analyte). Would this theoretically work? What would be a good blocking reagent to use? Thanks.