蛋白质微阵列生产用琼脂糖修饰玻片制备的条件优化

Optimizing of the Preparation Condition of Agarose Modified Glass Slide for the Production of Protein Microarrays

目的:建立一种以琼脂糖修饰的玻片为载体的蛋白质微阵列制备的优化方法,比较琼脂糖修饰玻片和醛基修饰玻片及氨基修饰玻片对蛋白质固定效率的优劣。方法:将羊IgG固定在载体表面,经过洗涤、封闭,再加入Cy3标记的兔抗羊IgG,孵育,洗涤后用共聚焦激光扫描仪获取图像,检测各点的荧光强度,根据荧光强度确定最佳琼脂糖浓度,最佳NaIO4浓度,最佳固定时间以及封闭时间等实验条件。结果:琼脂糖浓度为1.2％、NaIO4浓度为20mmol／L、固定时间为1h、孵育时间为45min时,蛋白质在载体上的固定效率和反应活性最高。在固定的抗体浓度相同的情况下,琼脂糖修饰玻片荧光强度是醛基修饰玻片的2.6倍,是氨基修饰玻片的9倍。结论:确立了蛋白质微阵列生产用琼脂糖修饰玻片制备的优化条件,用该优化条件制备的琼脂糖玻片更适合用于蛋白质微阵列载体。

Objective: To establish an optimized method for preparing protein microarray using agarose film coated glass slides and compare its protein loading capacity with aldehyde glass slide and amino glass slide. Method: Goat IgG was immobilized onto the surface of the substrates by an arrayer. After incubation, substrates immobilized with goat IgG were washed with PBS(pH 7.4) and blocked with 1% bovine serum albumin(BSA)/PBS(pH 7.4) . Substrates were then incubated with Cy3-conjugated anti-goat IgG. After wash with PBS (three times, 5min each), the slides were scanned by a laser confocal scanner. The optimal agarose concentration, NaIO4 concentration, immobilizing time, and incubating time were determined by average spot signal intensity. The protein loading capacity and linearity of the protein microarry spotted on three different substrates were investigated and compared. Result: When the concentrations of agarose and NaIO4 were 1.2% and 20ml/L respectively and the immobilizing time was one hour, and the incubating time was 45 minutes, the agarose coated glass slides based protein microarray performed 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 had. Conclusion: The optimized conditions have been established and the agarose film coated glass slide is more suitable for protein microarray preparation.