Covalent oriented coupling

1)  Couple your capture antibody
2)  flow over your his-tagged protein
3)  Mix the NHS/EDC reagents yourself (I use the same NHS and EDC aliquots
that I also use for activating chip surfaces so you don't have to make
those separately, just pull some aliquots from the freezer).
4)  Inject the NHS/EDC over the flow-cell(s) of interest.
5)  Immidiately inject the ethanolamine (or whatever primary amine
quencher you like) after the NHS/EDC mix.  I usually put the ethanolamine
in the rack and issue the quickinject command while the NHS/EDC activation
is happening so that the quench happens as soon as possible after the
activation.

That's the basic premise.  THe main variable then is how long to inject
the NHS/EDC solution.  The optimal injection time will balance two
parameters. The first is surface stability, and the second is surface
activity.  Too short an injection and you'll get marginal stabilization,
whereas long injections usually give you very stable surfaces.  Short
injections rarely affect the activity, but longer ones sometimes will.
Ideally you know something about the interaction already from measurements
on more unstable surfaces (KD, and ideally the off-rate, even if it's
collected on a decaying surface, so you have a reference).

Couple antibody to all four flow cells.  Sequentially step through the
flow cells, one by one, injecting the his-tagged protein and blocking.
Do this for three flow cells.  In each subsequent flow-cell activated for
a shorter period of time.  The experiment that worked for TfR was:

FC1:  60 second
FC2:  30 seconds
FC3:  15 seconds
FC4:  Don't do anything

Start a sensorgram over all 4 flow cells at 10 ul/min and let it run for
one hour.

Process the data and look at the drift over time.  There will be two
indicators of drift.  One will be the loss of material from the flow
cells, and the other will be the accumulation of material on the 4th flow
cell, captured out of what was lost on the previous cells.

Now, setup an analyte concentration series and run it three times (ideally
the same dilutions aliquoted into three tubes, or use the resealable caps
so the machine can use the same tubes over and over.  Those are a new
product out last year).  Determine KD and hopefully some kinetics for each
of those runs separately to give yourself an idea of the experimental
noise (you could globally fit the three runs together too).  Ideally
they'll all be the same for the runs and the flow cells.  However, what
you'll likely see is that the flow cell coupled for the longest time has
taken a hit in KD or in off rate.  For TfR, the HFE off-rate increased for
activations lasting longer than 45 seconds.  The increase was, at most,
2-fold, but it was reproducible, so I set the HFE activation around 30
seconds, which also gave stable surfaces.

Even after this procedure sometimes it's good to let the chip 'rest' for
an hour or two before beginning the experiment to let it stabilize.  It
also helps to run startup cycles (blank buffer injections, 5-10 of them,
before the experiment even begins) to let everything, including the
robotics, to settle down before beginning your samples.