I am looking to attach "Avidin" to APTES coated silicon surface without using biotin bonding. What I have in mind is to use "EDC or EDAC + NHS + avidin solution" and immerse the APTES coated surface in that. Eventually I want to have a biotinylated lectin binding to the avidin conjugated on the surface.
Comments, tips and references are welcome.
Thank you
Yes. You can use another chemical methods to bind Avidin on APTES.
But, Biotin - Avidin can bind up to four molecules of biotin simultaneously with a high degree of affinity and specificity.
Yes, you can use EDC/NHS to couple Avidin to the surface using the accessible COOH groups of the protein (there are plenty of protocols available, we typical use 0.1M NHS and 0.4M EDC. Note that the pH is crucial. EDC crosslinking is most efficient in acidic (pH 4.5) conditions and must be performed in buffers devoid of extraneous carboxyls and amines. MES buffer (4-morpholinoethanesulfonic acid) is a suitable carbodiimide reaction buffer. Phosphate buffers and neutral pH (up to 7.2) conditions are compatible with the reaction chemistry, albeit with lower efficiency. The pH is really important; at low pH, the amino group is protonated, and no modification takes place and at higher-than-optimal pH, hydrolysis of NHS ester is quick, and modification yield diminishes. Also make sure that your protein is stable in the used buffer!)
Another alternative could be coupling Avidin via glutaraldehyde to your surface using the accessible NH2 groups of the protein..
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Dear Ahmad,
you can also think about converting your amino functional surface with other suitable reagents.
disuccinimidyl carbonate (DSC): Morpurgo, M., et al. (1999). "N-hydroxysuccinimide carbonates and carbamates are useful reactive reagents for coupling ligands to lysines on proteins." J Biochem Biophys Methods 38(1): 17-28.
Also this article could be very helpful for you: Jonkheijm, P., et al. (2008). "Chemical Strategies for Generating Protein Biochips." Angewandte Chemie International Edition 47(50): 9618-9647.
I am just wondering if you are not generating big undefined aggregates of avidin if you try to activate the COOH groups of the protein. Should it not self-react with the lysine groups of other avidins? Maybe Anke can state on this.
Regards Volker
Well, Volker is right, if the conditions are not carfully optimized you will get more than a nicely defined monolayer of avidin on the surface. However, even a multilayer of avidin will bind biotinylated lectin ...
DSC would help in this regard since you can first convert your APTES surface with DSC to create a 'lysine reactive' surface on which you couple the avidin...
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