Universal-BIND™ Protocol


Introduction


The Costar® Universal-BIND™ surface is intended to covalently immobilize biomolecules via an abstractable hydrogen using UV illumination resulting in a carbon-carbon bond. Although the linkage is non-specific and does not allow for site-directed orientation of a biomolecule, this surface is very useful for the following applications:

1) immobilization of double stranded DNA,
 
2) immobilization of antigens of unknown structure (available functional groups unidentified),
 
3) immobilization of samples containing a mixture of biomolecules; ie. cell lysate samples, and
 
4) immobilization of other non-proteinaceous molecules, such as glycolipids.
This surface is available in both a 96 well plate catalog number 2503 and as a strip well plate.
 


UV Intensity Verification


In order to determine if the UV source to be used emits the necessary wave length of UV light, it is necessary to evaluate each new UV light source using the UV/EB Intensity Labels supplied with the Universal-BIND™ Plates or Strips.

1)  Use the BLANK plate supplied with the Universal-BIND™ Plates or Strips to determine if the UV light source to be used is of the proper wavelength and intensity and to determine the exposure time. Attach a UV/EB label to the top center of the BLANK plate (over wells D6, D7, E6, E7).
 
2)  Expose the plate to the UV light for 30 seconds.
 
3)  Compare the color of the UV/EB label to the CALIBRATION STANDARDS supplied. *Do not expose the CALIBRATION STANDARDS to UV light from windows or fluorescent room fixtures. This will change the color of the label.* If the CALIBRATION STANDARDS are not available or if they have been exposed to extraneous UV light, the optimal exposure time can be approximated by simply choosing the time at which the test label has turned a reddish-brown color. [Range: green (underexposure) - purple (overexposure)].
 
4)  If the 30 second exposure time is too short or too long, repeat steps 1-3 at different exposure times. *Be sure to attach a new UV/EB label for each exposure check.* **Increase or decrease exposure times by no more than 60 seconds at a time.**
 
5)  Record the optimal exposure time. This time will be used during the UV crosslinking step in the Covalent Coupling Procedure.
  


Covalent Coupling Procedure


1) Dilute biomolecule (SAMPLE) to be immobilized to 1-10 ug/mL in an appropriate buffer. (Phosphate Buffered Saline pH 7.4 and Carbonate Buffer pH 9.6 have been shown to be suitable buffers). DNA has been shown to bind more tenaciously at pH 3.0-5.0.
 
2) Add 100ul of diluted SAMPLE to each well. Be sure to leave a well blank as a control.
 
3) >Incubate for 1 hour at room temperature (RT)
 
4) Decant the solution. Expose the plate to UV light for the time determined to be optimal in the above UV Intensity Verification section.
 
5) Block the remaining active sites with a conventional blocking reagent - must be a protein blocker or equivalent; a detergent blocker is not sufficient (2% BSA has been shown to an adequate blocker for this surface). An incubation time of 30 minutes is recommended.
 
6) Decant the solution. Do not rinse.
 
7) Proceed with the remainder of the assay or procedure being performed. No special requirements exist.
Note: It has been shown that DNA remains bound after being subjected to the rigorous washing protocols associated with hybridization methods.
 


Performance Criteria

The Universal-BIND™ surface is tested for covalent immobilization using insulin, a small polypeptide that binds weakly via passive adsorption. The following assay is performed using two Universal-BIND™ plates. Insulin is allowed contact with the surface of both plates for 1 hour. One plate is then subjected to UV crosslinking; the other is not. Bound insulin is detected with a Horse Radish Peroxidase labeled Sheep anti-insulin antibody. UV mediated covalent immobilization of insulin is demonstrated in the following graph.

The results indicate a 2.8-fold increase in optical density for insulin that was UV crosslinked as compared to non-UV crosslinked to the Universal-BIND™ surface.
 


References:


Butler, John E. Immnochemistry of Solid-Phase Immunoassay. Boca Raton, Florida: CRC Press, Inc.: 1991.

Engvall, E. & Perlmann, P. Immunochemistry. 8:871: 1971.

Maggio, E.T. Enzyme-Immunoassay. Boca Raton, Florida: CRC Press, Inc.: 1980.

Pierce 1993 Immunotechnology Catalog & Handbook.

Tijssen, P. "Practice and Theory of Enzyme Immunoassays" in Laboratory Techniques in Biochemistry and Molecular Biology. Vol. 15. Ed. R.H. Burdon and P.H. van Knippenberg, New York: Elsevier Biomedical: 1985.

Wong, Shan S. Chemistry of Protein Conjugation and Crosslinking. Boca Raton, Florida: CRC Press, Inc.: 1991.