XENOBINDTM COVALENT BINDING PRODUCTS FOR PROTEIN & PEPTIDE IMMOBILIZATION ON MICROPLATES and GLASS
XENOBIND™ one-step covalent binding products provide a simple and rapid method for immobilizing proteins and peptides for use as capture probes in immunoassays. The advantages of covalent binding for immobilizing antibodies and peptides have been documented in the literature: higher binding capacity 1, the ability to bind small peptides which have no hydrophobic regions to attach passively to hydrophobic plates2, and elimination of the displacement of active antibodies either by serum proteins such as fibrinogen3 or by washing to remove unbound material during the assay procedure4.Introduction
XENOBIND plates are molded to our exacting specifications of optical clarity and uniformity. They are then treated by our proprietary liquid phase process to produce covalent binding sites. This liquid phase process allows control of the uniformity of the treatment to ensure a low coefficient of variation, unlike other processes which rely on random radiation or non-uniform plasma processes to produce active groups on the surface. The XENOBIND process eliminates edge effects. Since treatment is restricted to the surface, the plates have exceptional clarity and show none of the yellowing typical of other microwell plates.XENOBIND Clear Plates
XENOBIND White & Black Plates
XENOBIND plates are also available in two opaque versions: XENOBIND WHITE for chemiluminescent assays and XENOBIND BLACK for fluorescent assays. The white and black plates are treated by the same liquid phase process as the clear plates and have the same binding capacity per well. The opacity of the plates eliminates "crosstalk" between the wells, so that the emitted signal from one well does not interfere with the signal from an adjacent well. Typically, the sensitivity in a chemiluminescent or fluorescent based assay is 100 to 1000 times more sensitive than a colorimetric assay and approaches the sensitivity of a radioactive isotope-based assay without the handling and disposal problems of isotopes.
Using XENOBIND Plates
XENOBIND microwell plates require no activation step by the user to produce a covalent bond. The protein to be bound is simply dissolved in a buffer solution at a pH above the isoelectric point of the protein (important because the binding takes place through the amine groups on the protein, and these must be in the free form for binding to take place); the protein solution is then incubated in the wells for 2-3 hours at 37 degrees. This results in the formation of a covalent bond between the surface and the protein.
In most cases the binding capacity is substantially increased by covalent binding. For peptides this can be as much as 100-fold compared to passive binding2. For typical antibodies high binding levels are achieved at much lower concentrations than with passive binding5,6. This is the result of the irreversible nature of the covalent binding process; monolayer coverage can be achieved at low concentrations, limited only by the diffusion rate of the antibodies to the surface. Monolayer coverage is usually about 300 ng. per square centimeter, and it can be achieved easily with concentrations containing only twice that amount of protein in agitated systems. In most cases the presence of surfactant in a protein solution does not inhibit covalent binding with XENOBIND plates as it does with passive binding.
Return to Products Page Return to Home Page
GLASS MICROSCOPE SLIDES, COVERSLIPS and PARTICLES
The proprietary surface chemistry that XENOPORE has pioneered for the attachment of proteins and peptides to the surface of microwell plates and other plastics has been adapted to the activation of glass surfaces for the covalent attachment of proteins, peptides and other ligands. This new surface chemistry produces a high density of reactive groups which are then used to immobilize the ligand of choice. The density of ligand is approximately 1013 per square centimeter. As standard formats there are available 25 X 75 mm microscope slides and No. 1 1/2 cover slips, 22 X 40 mm. Other sizes and thicknesses are available as special orders.
In addition, this surface chemistry has been applied to glass and silica particles. These have a distinct advantage over plastic particles in the submicron range because their higher density makes it much easier to collect these particles by centrifugation in contrast to the almost neutrally buoyant polystyrene particles whose Brownian motion below 0.1 microns prevents collection in ordinary centrifuges.
Custom coating of any shape glass is also available. Please contact us to discuss your needs.
References:
1. DeBari, V.A., "The Binding of Proteins to Activated Polystyrene Surfaces", presented at AACC National Meeting, New Orleans, July, 1988
2. Katagiri, M., Kishimoto, K., Miyoshi, N., Sakamoto, Y., and Ishibashi, F., "Enzyme Immunoassay for Peptides with New Microwell Plates", Chem Express v. 5 #8 pp. 617-620 (Japanese) Chem Abstracts v. 113 168257q
3. Scott, C.F., "Mechanism of the Participation of the Contact System in the Vroman Effect. Review and Summary", J. Biometr. Sci., Polymer Ed. v. 2, pp173-181, 1991
4. Butler, J.E., Peterman, J.H., Suter, M., and Dierks, S.E., "The Immunochemistry of Solid Phase Sandwich Enzyme
-Linked Immunosorbent Assays", Fed Proc v. 45, pp 2548-2556, 19875. Douglas, A.S., and Burshteyn, A., "The Binding of IgG to Untreated and Chemically Treated Polystyrene Microwell Plates", Presented at CliniChem 86, New York City, Nov., 1986
6. LaSalle, M.D., Gentile, V.G., Pertussi, R., Keil, L. and DeBari, V.A., "The Enzyme-Linked Immunosorbent Assay of Antibodies to Vimentin in Patients with Systemic Lupus Erythematosus", J. Clinical Immunoassay v. 12 #2 pp111-114 1989
SUGGESTED BINDING PROCEDUREProtocol For Binding Peptides or Proteins to XENOBIND Plates
1. Prepare a solution of the peptide or protein in 0.05M phosphate or carbonate buffer at a pH which is above the isoelectric point of the peptide or protein. Peptide or protein concentration should be in the range of 1-3 ug/ml, although for some peptides, concentrations as high as 20 ug/ml may be required for complete coverage.
2.Fill each well of the plate or strip with 200 ul of peptide or protein solution.
3.Incubate for 2 hours at 37 C. Alternatively, incubate overnight at room temperature. The plates should not be incubated at 4 C since the reaction will not proceed effectively.
4. Empty wells and wash with 300 ul of 0.05M phosphate buffer pH 7.5 containing 0.15M NaCl (PBS) and 0.1% of Tween 20 or Triton X-100. Note: Peptide or protein solution can be pooled upon removal from wells. Protein concentration can be adjusted and the solution used in subsequent couplings.
5.Empty wells and add 250 ul of 3% bovine serum albumin (or powdered milk protein) prepared in PBS. Incubate for 2 hours at 37 C or overnight at room temperature.
6.Wash plates three times with 300 ul of PBS containing 0.1% Tween 20 or Triton.
7.Allow plates to air dry at room temperature. Prior to coupling, plates should be stored at room temperature in a sealed plastic bag and protected from alcohols and amine containing compounds. Following coupling, plates should be stored at 4 C. Under these conditions, most bound proteins will be more stable than the protein in solution.
Return to Products Page Return to Home Page
ORDERING INFORMATION
PLASTIC PRODUCTS
Catalog No. Description Quantity
XBP 005 00 Clear Plates 5 Plates
XBP 025 00 Clear Plates 25 Plates
XBP 050 00 Clear Plates 50 PlatesXBS 005 00 Clear Strips 5 Plates
XBS 025 00 Clear Strips 25 Plates
XBS 050 00 Clear Strips 50 PlatesWBP 005 00 White Plates 5 Plates
WBP 025 00 White Plates 25 Plates
WBP 050 00 White Plates 50 PlatesWBS 005 00 White Strips 5 Plates
WBS 025 00 White Strips 25 Plates
WBS 050 00 White Strips 50 PlatesBBP 005 00 Black Plates 5 Plates
BBP 025 00 Black Plates 25 Plates
BBP 050 00 Black Plates 50 PlatesBBS 005 00 Black Strips 5 Plates
BBS 025 00 Black Strips 25 Plates
BBS 050 00 Black Strips 50 PlatesXBB 001 00 XENOBEADS 100 Beads
XBB 005 00 XENOBEADS 500 BeadsUDB 005 00 Underivitized 500 Beads
GLASS PRODUCTS
MBC 000 10 Microscope Slides 10 Slides
MBC 000 50 Microscope Slides 50 Slides
MBC 001 00 Microscope Slides 100 SlidesVXB 000 10 Cover Slips 10 Pieces
VXB 000 50 Cover Slips 50 Pieces
VXB 001 00 Cover Slips 100 PiecesGCN 000 20 1 micron particles 20 mg.
GCN 001 00 1 micron particles 100 mg.
