Suggested Binding Protocols for XENOSLIDES
Suggested binding protocol for use with XENOSLIDE A (aminosilane) and aminosilane coverslips:
1. Slides are silanated and ready to use as received. They can be stored at room temperature.
2. Prepare a solution of the cDNA to be spotted. The DNA concentration can be in the range of 1 ng to 1 ug per ml.
3. Spot size can be controlled by use of solvent mixtures. Correct choice of co-solvent will result in lower surface tension of the mixture compared to water and controlled spreading of the spot. Volatility of the solvent mixture and thus the drying time can also be controlled by solvent composition. Use of a lower volatility co- solvent will increase the drying time. DMSO is frequently use since it is a good solvent for DNA, is miscible with water in all proportions and has lower surface tension and lower volatility than water. Typically, up to 50% DMSO is used. Alternatively, glycerol can be used in place of DMSO.
4. Spot the solution onto the slide. If you are using water only, it is helpful to maintain a humidity of 75-80% for a few minutes to allow binding to take place.
5. Some researchers crosslink the DNA to the slide by exposing the slide to UV light up to about 200 millijoules of radiation. The slide is now ready for hybridization.
Note: For additional binding suggestions, see the recent article from TIGR in Biotechniques magazine, Sept. 2000.
Suggested Binding Protocol for XENOSLIDE N (nickel chelate surface) and nickel chelate cover slips. Also for use with cobalt chelate surfaces.
1. The slides are coated with the chelating agent and are charged with either nickel or cobalt ions. They are ready to use as received. They can be stored at room temperature.
2. Prepare a solution of the his-tagged protein or peptide in a neutral pH buffer or a slightly basic buffer. Tris buffer is a good choice. The solution concentration should be in the range of 1 - 3 ug/ml.
3. Spot the solution onto the slide or cover slip. Keep the slide wet for 5 -10 minutes by putting it into a humid chamber. This allows the binding to take place.
4. Air dry the slide. It is now ready for use in capture experiments.
Suggested binding Protocol for XENOSLIDE D (aldehyde surface)
1. The slides are chemically modified to have a high density of aldehyde groups on the surface. These groups immobilize aminolabeled DNA, proteins and peptides by Schiff base chemistry.
2. Prepare a solution of the amino labeled oligo in a neutral pH buffer at a concentration of 1 -3 ug/ml. Do not use Tris buffers because these contain free amine groups that can react with the plate surface and prevent oligo binding.
3. Spot the oligo onto the slide. Keep the slide wet for 5 -10 minutes by putting it into a humid chamber at a relative humidity of 50 - 70 %.
4. The Schiff base reaction is reversible at acid pH. For greater stability, reduce the Schiff base with sodium borohydride. A 1% solution in water, 30 minutes at room temperature is usually adequate. This also blocks unreacted groups. If you omit the reducing step, block the unreacted aldehyde groups with a solution of ethanolamine in water (1% by volume).
5. Air dry the slide. It is now ready to use in hybridization tests.
Suggested binding Protocol for XENOSLIDE E (epoxy surface)
1. The slides are chemically modified to have a high density of epoxy groups on the surface. The epoxy group is highly reactive to primary amino groups and hydroxy groups at high pH.
2. Prepare a solution of the oligo or peptide to be bound in a solution at pH 10.5 - 11 at a concentration of 1 - 3 ug/ml
3. Spot the oligo solution onto the slide. Keep the slide wet for 5 - 10 minutes by putting it into a humid chamber at relative humidity of 50 - 70%.
4. Blocking of the slide is usually not necessary because the reaction of the epoxy group at neutral pH is quite slow. Therefore DNA and oligos will only bind by hybridization.
5. Air dry the slide. It is now ready for hybridization experiments.
Suggested Binding Protocol for XENOSLIDE S (streptavidin surface)
1. The slides have a high density of streptavidin immobilized on the surface. The binding capacity is approximately 5 picomoles of biotin binding sites per cm. sq. The slides are ready to use as received. Store unused slides in their container in the refrigerator.
2. Prepare a solution of the biotinylated oligo at a concentration of 1 - 3 ug/ml in a neutral pH buffer such as 1 X SSC or phosphate buffer.
3. Spot the oligo solution onto the slide. Keep the slide wet for 5 -10 minutes by putting it into a humid chamber at a relative humidity of 50 -70 %
4. Air dry the slide. It is now ready for hybridization experiments.
