基于In-Cell Western对Ryanodine受体2检测方法的建立

The Development of In-Cell Western Assay in Detecting Ryanodine Receptor 2

In-Cell Western技术是基于近红外激光成像系统而开发的检测技术,可应用于大分子ryanodine受体2(Ry R2)蛋白的检测。本研究通过对In-Cell Western固定剂的选择、细胞通透化、封闭液的选择、抗体工作浓度和心肌细胞密度等方面进行优化,建立心肌细胞Ry R2蛋白检测的InCell Western方法,并分别用SGF和Verapamil以及免疫细胞化学技术对研究结果及方法进行了验证。优化后的In-Cell Western参数:H9C2细胞的种板密度选择1×104个/孔,选择甲醛作为固定剂,细胞经过Triton X-100洗脱液通透化处理,选择酪蛋白作为封闭液,选择1∶500稀释的Ry R2一抗工作浓度。应用优化的参数检测心肌细胞Ry R2蛋白,并用前期已被证明能显著提高心肌细胞Ry R2蛋白荧光值的土茯苓黄酮和Verapamil进行验证,同时与免疫组化方法检测心肌细胞Ry R2蛋白相比,检测结果一致。表明本文建立的In-Cell Western方法检测大分子功能蛋白Ry R2是可行的。

In-Cell Western is a detection technology using a near infrared laser imaging system. We optimized the parameters of an In-Cell Western assay for the detection of ryanodine receptor 2（ Ry R2）proteins. The protocol optimization included,H9C2 cell density was 1 × 104 cell/well; formaldehyde was selected as a fixing agent; the cells were treated with Triton X-100; casein was selected as the blocking reagent; and the Ry R2 antibody concentration was 1 ∶ 500. In-Cell Western was tested to detect the expression of Ry R2 protein in H9C2 after treating with SGF and Verapamil. The results were consistent with immunocytochemistry in paired samples. The results showed that the In-Cell Western assay could be applied for the detection of Ry R2 protein.