Optimizing Of The Preparation Condition Of Agarose Modified Glass Slide For The Production Of Protein Microarrays

OBJECTIVE Efficient immobilization of biomolecules is a key factor in determining the overall success of a protein microarray. Most of the microarrays are based on surfaces and formats that have been used for DNA arrays. Glass slides coated by epoxy- or aldehyde-terminated alkylsilanes or by polylysine can also be used to immobilize proteins, antibodies and antigens. But protein immobilezed on these surfaces turns to denature. Agarose film has a three-demensional structure with tiny pores in it. This porous structure enables agarose film to hold much more protein molecules and water as well. So agarose film coated glass slides not only have a big protein loading capacity but can minimize the denaturation of the immobilized protein. This study is to establish an optimized method for preparing protein microarray using agarose film coated glass slides and compare its protein immobilization efficiency with aldehyde glass slide and amino glass slide.METHODS Clean glass slides are coated with different concentration of agarose film. Goat IgG was immobilized onto the surface of the agarose modified glass slide by a microarray arrayer. After washing off the unbound protein and blocking the reactive group on the surfaces, Cy3-conjugated anti-goat IgG was added. After incubating, wash again before the slides were scanned with laser confocal scanner. The optimal agarose concentration, NaIO4 concentration,immobilizing time, incubating time were determined by average spot signal intensities. The protein loading capacity of the protein microarry spotted on three different substrates were investigated and compared.RESULTS When the concentration of agarose and NaIO4 was 1.2% and 20ml/L respectively and the immobilizing time was one hour, the incubating time was 45 minutes the agarose coated glass slides based protein microarray performed the best. And it had a 2.6 fold and 9 fold higher protein loading capacity than aldehyde glass slides and amino glass slides based microarrays.CONCLUSION The optimized conditions has been established and the agarose film coated glass slides are more suitable for protein microarray preparation.