questions with NTA chip

Hi Jane,
This is a good improvement of the interaction. Lowering the ligand concentration will make the kinetics better but the bulk contribution is coming from the analyte so that may not change. If possible you can incorporate the RI in the model and see if the fit if better. Please check if the fitted RI is the same as the observed since fittings can overcompensate to make the fit better. As an alternative you can set a value to the RI as a constant. I would suggest making the association a bit longer. I would go to 3 minutes - if possible to 5 - to get more curvature in the lines.
One concern about the highest concentrations where the binding seems to linear/absent. What is your highest concentration analyte? It seems that there non-specific interaction becomes a problem.
Arnoud

Thanks for your help.
The highest concentration was 100uM.
The other issue that bothered me is false high Rmax. RU was 600, ligand MW 108KDa, the analyte is 520Da. I also have a difficult time regenerating the chip. The baseline is getting higher after every cycle.
What do you think the problem?
Thanks.

Jane

Hi,
Rmax is often calculated wrong when there is not enough curvature in the graphs or when there is a high bulk contribution. You can calculate the theoretical Rmax from the ligand and analyte size and the amount of ligand captured. You can also start the fit by fixing the Rmax and first determine the kinetic parameters and then at the end float the Rmax again. Start with fitting the dissociation to get the k_d. Etc.
A higher baseline is an indication that either the ligand or the analyte is sticking to the sensor surface. On the other hand when all channels have a comparable increase it can be drift in the system or just a slightly different flow buffer solution. When the capture of the ligand is not diminishing I would not worry too much.

You can use some harsher solutions to try to wash away what has bound over time. I would suggest you try a 1/6 - 1/3 Ionic solution ( www.sprpages.nl/kinetics/regeneration ) in some short pulses (~10s) or 0.85% H₃PO₄ as an acidic solution. Best way to regenerate a sensor surface is to start with the mildest option, use short pulses and wash until a stable baseline.
Arnoud

Hi Arnoud,
I am still struggling with my SPR measurement, I simply cannot repeat the results.
As I mentioned last time, I noticed that the baseline increased (only active channels FC2 and FC4) and the ligand capture level decreased after each cycle. Those observations made me believe the regeneration with 350mM EDTA was not complete, and some proteins stuck on the sensor (that's why I chose using single-cycle mode). I cleaned the sensor with Imidazole, NaOH, glycine-HCl (pH2), and Urea, but it got worse (see the attached figure of some sensorgrams). I have not tried some other harsh conditions yet, just afraid it might get even worse. Do you know what's wrong with it?
Thanks.

Jane

OK, let's go back and take a single step at the time.
You are using a NTA sensor chip which means that a sensor surface with dextran is modified with the NTA-moiety (assuming you use the Biacore). This means that in the ground this is a dextran chip. The NTA is loaded with Nickel so it can bind the 6xHIS protein sequence. The first thing is to check if the loading of the Nickel is much lower now than when you started using the chip. It is a good idea to note the nickel loading each time so you can monitor the integrity of the surface. It is normal that the loading wil drop the first few Ni-loading / EDTA regeneration cycles and then it will drop slowly. As with all surfaces, they do not live forever.

When you decide that the Ni-loading is still ok then look at the 6xHIS protein loading in the same way as the Ni-loading.

Before you decide to inject the compound you can do some test for non-specific binding to try to pinpoint if the compound is raising the baseline. First strip the surface to base NTA and inject the compound and record the possible non-specific binding. Ideally it should not bind and baseline should return without regeneration. The load the nickel and repeat etc.
In this way you have to find out what is causing the problem and optimise the experiment.

What is a higher baseline? Are we talking about 10 RU or more than 100 each cycle?
I would not try to get the last RU of the surface since this will lower the life-time of your chip. Regeneration solutions can lower or raise the baseline of the surface and this can be different between channels due to the type of protein and amount of protein loading. Only extensive washing can bring the baseline back provided that there were no irreversible changes to the surface.

I know that sensor chips are fairly expensive but sometimes it is better to sit back and start over fresh.

Note: you may explore the BIA-pages ( www.biapages.nl ) for complementary assays to confirm your finding.

Kind regards
Arnoud