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Covalent (Crosslinking) Immunoassay Plates
Covalent coupling surfaces for smaller biomolecules
We offer a variety of functionally-activated microplates for covalent attachment of peptides, nucleic acids, haptens, and other biomolecules for ELISA and other plate-based assay techniques. These plates bind with functional groups such as amino (NH2), sulfhydryl (SH), carboxyl (COOH), and phosphate (PO4).
Covalent coupling is based on the formation of a single covalent bond between the polymer surface and the biomolecule. Small biomolecules can be immobilized using this technique, as can medium and large molecules that possess the appropriate functional group(s). Since coupling occurs via specific functional groups, biomolecular orientation can also be manipulated by the user.
| Biomolecule | Surface type | Key features |
|---|---|---|
| Biomolecules with free NH2 and/or SH groups e.g. proteins, peptides, aminated oligos | Nunc Immobilizer Amino | Ideal if your biomolecule does not bind well to a passive surface and it possesses one or more free primary amino or sulfhydryl groups (peptides, oligonucleotides, proteins, proteoglycans) |
| Amine-containing peptides and other molecules | Pierce Maleic Anhydride | Useful for immobilizing peptides and other ligands that do not coat efficiently by passive adsorption. Reaction of the maleic anhydride groups with primary amines (-NH2) results in formation of amide bonds that are stable at neutral pH and above |
| Sulfhydryl-containing molecules | Pierce Maleimide | Ideal for binding sulfhydryl-containing molecules that are difficult to coat onto polystyrene plates, such as peptides that contain a terminal cysteine. An especially useful tool for assessing specific anti-hapten antibody titers during antibody production. |
| Biomolecules with -COOH or -PO4 groups | Nunc CovaLink | Designed to facilitate the coupling of molecules bearing a free carboxyl or phosphate group. Therefore,peptides, haptens, and DNA can be coupled. |
| 5' phosphorylated or 5' aminated oligonucleotides and nucleic acids | Nunc NucleoLink | Ideal for binding nucleotides and for hybridization assays |
Covalent plate types
Nunc Immobilizer Amino surface
The Immobilizer™ Amino surface forms stable covalent bonds between its electrophilic groups and the biomolecule's free amino acids or sulfhydryl groups. Using its unique spacer arm chemistry, the surface provides extremely low non-specific binding to improve assay sensitivity. With no need for an ancillary coupling agent, this surface can simplify your assay development by eliminating the need for a time-consuming blocking step.
The Immobilizer Amino surface is ideal:
- If your biomolecule does not bind well to a passive surface and it possesses one or more free primary amino or sulfhydryl groups (peptides, oligonucleotides, proteins, proteoglycans)
- To obtain a highly sensitive assay with excellent reproducibility and low background
- To minimize the use of a coating reagent
- To reduce the number of steps required to prepare plates
- To avoid unwanted reactivity associated with a blocking reagent
Pierce Maleic Anhydride
Pierce Maleic Anhydride Activated Plates allow attachment of amine-containing peptides and other molecules to microplate wells for use in binding assays.
Features of Maleic Anhydride Activated Plates:
- Spontaneously react with primary amines
- A direct, simple approach to covalently attach molecules to the well surface of an EIA plate
- Maleic anhydride maintains its integrity and availability for coupling for months under dry, room temperature conditions
These plates are useful for immobilizing peptides and other ligands that do not coat efficiently by passive adsorption. Reaction of the maleic anhydride groups with primary amines (-NH2) results in formation of amide bonds that are stable at neutral pH and above. Acidic conditions hydrolyze the bond; for example, the half-life of hydrolysis at pH 3.5 and 37°C is approximately 11 hours. Therefore, coating reactions are best performed at pH 8–9, and the reacted plates are best used at pH > 7 for ELISA and other methods.
Pierce Maleimide
Pierce Maleimide Activated Plates are ideal for binding sulfhydryl-containing molecules that are difficult to coat onto polystyrene plates, such as peptides that contain a terminal cysteine. Our coated plates are an especially useful tool for assessing specific anti-hapten antibody titers during antibody production.
Features of Maleimide Activated Plates:
- Pre-blocked to reduce nonspecific binding
- Easy (spontaneous) immobilization of peptides derivatized with a terminal cysteine and proteins with free sulfhydryl
- Binding capacity: 100 to 150 pmol of a sulfhydryl-containing peptide (307 Da) per well
- Activation level: 100µL
Maleimides react with free sulfhydryl group(s) at pH of 6.5–7.5, forming stable thioether linkages. Reaction with amines becomes significant at pH > 7.5. Some sulfhydryl-containing peptides and proteins may oxidize in solution and form disulfide bonds, which cannot react with maleimides. Disulfide bonds can be reduced to produce free sulfhydryls. The TCEP Disulfide Reducing Gel enables peptide or protein reduction while recovering the sample in the absence of reducing agents. Sulfhydryls can be introduced via amine modification using N-succinimidyl S-acetylthioacetate (SATA) or 2-iminothiolane-HCl (Traut's Reagent).
Nunc CovaLink
The CovaLink surface is designed to facilitate the coupling of molecules bearing a free carboxyl or phosphate group. Therefore, peptides, haptens, and DNA can be coupled. The surface uses a spacer arm to increase accessibly, thereby enhancing overall surface reactivity.
Nunc NucleoLink
Nunc NucleoLink Breakable Modules are ideal for hybridization assays due to the heat resistant resin. The unique design offers error-free flexibility; each well locks in the frame and maintains the same height to provide accurate reading and efficient washing.
- Use in place of time-consuming methods such as gel electrophoresis and Southern blotting
- Usable with any “ELISA-like” detection and measurement method
- Compatible with standard microplate readers
- Consists of 1 x 8 strips to be fitted into a 96-well format frame
- V-shaped wells with flat, readable bottoms and thin walls (0.35mm)
- Withstand temperatures from –20° to +120°C
- Working volume: Strips 100μL
Related categories
Crosslinking, modifying, and labeling proteins are commonly used techniques for studying the structure and interaction of proteins. Crosslinking is the process of chemically joining two or more molecules by a covalent bond.
Modification involves attaching or cleaving chemical groups to alter the solubility or other properties of the original molecule. Labeling generally refers to any form of crosslinking or modification whose purpose is to attach a chemical group (e.g., a fluorescent molecule) to aid in detection.
FAQs
A selection of FAQs is listed below. For more technical information and FAQs, please visit our Solid Phase Guide ›
The Immobilizer surface binding has several advantages over passive binding to solid phases. One of advantages of 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 surfactant will further avoid unspecific binding in the wells. The coefficient of variation (CV) between the individual wells will also be very low and will give uniform, reproducible results.
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.
CovaLink NH Modules are surface modified optically clear polystyrene modules in strips of eight. They allow covalent binding of distinct groups of proteins, peptides, oligosaccharides, and DNA. This covalent binding feature allows orientation of the bound molecules so that the active site of the molecule is available for biochemical activity. A key feature of the CovaLink is that the polystyrene surface is grafted with secondary amino groups which serve as bridges for covalent binding. The optically clear surface allows reading of fluorescent or colorimetric signals.
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