FAQ:
Thermo Scientific Nunc Immobilizer™ Amino plates and strips
What is the technology behind the Immobilizer plates and strips?
The technology is based on the unique anthraquinone photo-coupling method which provides a simple one-step procedure for covalently coupling bio-molecules. The photoprobe consists of 3 parts: The anthraquinone molecule, the ethylene glycol spacer and an electrophilic group. The density of the electrophilic groups and the spacer design is optimized for immobilizing either peptides, proteins, or antibodies.
Which type of plates and strips should I use for the different detection methods?
We recommend using transparent polystyrene plates and strips for colorimetric assays, white polystyrene plates for bio and chemiluminescence assays, and black polystyrene plates for fluorescence assays.
Why is the Immobilizer better than passive binding?
The Immobilizer binding has several advantages over passive binding to solid phases. One of advantages for the Immobilizer plates and strips is the strong covalent binding of the anthraquinone to the microwell plate. This means that there will be no leaching of the bound molecule. The use of stringent washing procedures and TWEEN® 20 will further avoid unspecific binding in the wells. The coefficient of variation (CV) between the individual wells will also be very low, will give uniform and reproducible results.
Should I optimise the concentration of the target molecule?
Yes. We recommend that the amount of target molecule is optimised. Initially, we suggest making a titration of the biomolecule and load different concentrations in the wells in the dedicated buffer.
Which detection method can measure low amounts of analyte?
Low concentrations can frequently be detected using the Immobilizer™ plates and strips. A low limit for detecting a good signal is normally around OD=1.0. Depending on enzyme, substrate, equipment, and reaction requirements the different detection assays can be optimised to meet the necessary sensitivity. In the theory luminescence assays will be the best choice for detecting low signals.
Could there be any problems during the coupling procedure?
To avoid any problems ensure that the biomolecule has a free primary amine, thiol or hydroxyl group that will react with the electrophilic group on the spacer. Also, avoid competition from other nucleophiles like ethanolamine, lysine, or TRIS during the coupling. Further, do not use non-ionic detergents like TWEEN® 20 in the coupling buffer since the covalent coupling of the bio-molecule will be suppressed.
Why do we advise TWEEN® 20 in washing and in assay buffers?
TWEEN® 20 blocks the binding sites that not are bound. In combination with the stringent washing procedure this detergent generally improves the signal to noise ratio of the assay. We recommend using buffers containing 0.05-2% TWEEN® 20.
Can the molecule be bound but not recognised during the detection?
Yes. If lysine or cysteine is a part of the epitope recognised by the antibody then a binding of these amino acids to the photoprobe will lead to a reduced recognition and a lower signal. An increase in the peptide concentration could solve this problem.
For how long time should I incubate the target molecule to the Immobilizer?
Coupling of the target molecule to the anthraquinone photoprobe proceeds very fast. Results shows that incubation with gentle agitation for 2 hours at room temperature secures that the available active sites are saturated.
For how long are the plates stable?
The Amino Immobilizer plates and strips are extremely stable for a long time if stored at room temperature protected from light. In contrast, plates for passive adsorption have a relatively short shelf life. Nunc guarantees a 24 month shelf life after the date of manufacturing.
Can I use target molecules other than peptides, and proteins?
Yes, but the protocol needs to be optimised and the target molecule should be modified if it does not contain nucleophilic groups.
|
