Forum :: Immobilization with mPEG-NH2 in stead of EtOH-NH2


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This forum is intended for questions about kinetics, Surface Plasmon Resonance and the instruments related to these techniques.

TOPIC: Immobilization with mPEG-NH2 in stead of EtOH-NH2

Immobilization with mPEG-NH2 in stead of EtOH-NH2 04 Mar 2014 15:49 #1

Hello,

I am running some experiments and I experience non-specific interaction with my reference flow cells, that are blocked with ethanolamine from the Biacore Immobilization kit. I would like to try a different blocking approach, by blocking both the active and reference flow cells with mPEG-NH2 of 350 Da.

I was wondering I anyone of you has experience with this and can give me some advice in what buffer and at which pH immobilization of mPEG-NH2 is the most efficient.

Many thanks in advance!

J

Immobilization with mPEG-NH2 in stead of EtOH-NH2 07 Mar 2014 23:00 #2

Hi J,

The 1 M ethanolamine blocking is at pH 8.0 and serves two purposes.
1) deactivating active groups by binding ethanol amine.
2) flush out electrostatically bound ligand by high salt (1 M) and high pH.

I understand that you use a 'deactivated' channel as reference. It can be benificial to immobilize a non-related protein such as an IgG or BSA.

For the mPEG-NH2 I would choose a pH 7.5 - 8.0 and try to use a high concentration (> 0.25 M) if possible. Probably i would add a high salt (1 M NaCl) after that.

Let us know what you did.

kind regards

Arnoud
The following user(s) said Thank You: JVanb

Immobilization with mPEG-NH2 in stead of EtOH-NH2 20 Mar 2014 09:44 #3

Hello,

I tested blocking with mPEG-NH2 by dissolving a high concentration of mPEG-NH2 in different buffers, Na-acetate pH 5,5; HBS pH 7.4 and carbonate buffer pH 9. 600s injection of mPEG-NH2, diluted 1:10 directly out of stock in HBS pH 7.4 , resulted in blocking of both reference and active flow cells.

Since I used a low molecular weight PEG (350Da) I did not see a significant RU gain after deactivation (in analogy with ethanolamine). I tested this blocking by injecting high antibody concentrations on the activated flow cell after the blocking with mPEG-NH2, and I did not have any covalent blocking of the mABs, which proves the activated surface was deactivated sufficiently.

This allowed me to perform kinetic analysis with mABs in solution and immobilized antigen, whereas the ethanolamine blocking did not make this kind of set-up possible, due to high non-specific binding of different mABs to the ethanolamine reference.

kind regards!

J
The following user(s) said Thank You: Arnoud, Food Chemist
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