Biacore - What is happening? (Mar/29/2006 )

Hi all,

I have been managing some problems regarding my biacore problems. Yesterday I found the following:

I treat a CM5 chip with NHS:EDC (1:1) for 7 minutes then with ethanolamine 1M pH 8.5 also for 7 minutes (no protein immobilization has been taking place, only NHS:EDC and ethanolamine). After that I show non specific binding when injecting any analyte over the treated channel. It seems like an analyte retention. If the channel has not been treated, we have no non specific binding, non analyte retention (so it is not a dextran related problem).

What is happening?

Hello,

I don´t undertand well what you are trying to do. You´re activating the surface with NHS:EDC, but are blocking all the reactive groups with ethanolamine, so when you inject an analyte, you´ll only see the non-specific binding between the analyte and the chip surface.

When you say 'injecting ANY analyte', what do you mean?...what analytes have you used?....Can you say any more?

This is a long troubleshooting history.

Five months ago, we performed routinely experiments with no problem. At any time, we start to see rare signals, like a non specific binding.

We did not understand these rare signals if never had appeared.

Last monday, and after tedious checkings, in order to know if the reason is related with the immobilization process I performed what I said in my previous message. It does not matter what is the analyte. This problem is analyte-independent

Perhaps am I bloking unefficiently the active groups? but why?

I do not know

When you say that perhaps you are blocking unefficiently the active groups....Do you mean the active groups in the ligand molecule? I suposse that the ligand is a protein that you immobilize by the amine coupling method on the carboxyl dextran suface of the CM5


Or do you think that you are blocking the active groups in the analyte molecule?...Is the analyte another protein?

Do you think that there may be a mass transport problem on the chip surface?

Does it happen in different experiments? different ligand-analyte pairs?...different concentrations?...

I refer to the active ster gropus formed when treated the carboxilate groups with NHS:EDC. I want to say that may be the ethanolamine is not deactivating these activated groups. Also I have though about a machine problem. But System Check give well parametres

-Are there other users of the equipment?...Are they having any problem like yours?...

-How about cleaning and maintenance protocols?.....Who is the person that does the maintenance of the equipment?....I think that the most important thing for an automatic instrument is the cleaning and the maintenance....preventive maintenance, preventive cleaning...


-It seems that the problem appears when you inject the analyte...have you tried injecting the analyte buffer without the analyte?


-You think that a possibility is that the ethanolamine isn´t be able to deactivate all the activate groups .....in that case you would have two options: increase the quantity of ethanolamine (concentration, time), or decrease the quantity of activate groups......but for me is difficult to believe that because I suposse that ethanolamine is calculate to be in excess, but I don´t know...

i also think that you need to substract the response of the analyte-buffer w/o analyte to obtain the real binding curve of your analyte

i don't know why, but the refractive index of the injected solutions w or w/o the analyte give a detectable response on your sham (ligand)-immobilized Fc...
have you normalize the signal response before experiment?
i hope your EDC and NHS solutions are ok (not out of date, clean and stored at -20C), since if the 1:1 ratio is not respected, the CM would be modified in a such a way that some non-specific interactions could occur, especially with the positive charges of the EDC...
we can find a lot of reasons that could explain your observations, but this need some more controls

Seb

Ok, thank you very much for your suggestions and help.

More:

- When injecting buffer the signal is not very stable. It seems like it increases slowly as the injection time proceeds.

- I am only the person which works with the Biacore. The routinely maintenance protocolos are followed: Desorb and removal of salt weekly, sanitize monthly.

- NHS, EDC and ethanolamine are stored at - 20ºC in 0.5 mL alicuots.

- Next thursday I am going to try our experiments in another Biacore.

What a rare problem!!!

Hello,

when you did your experiment, was the signal similar to any of the images in figure 16 in this review?



Good luck next Thursday!