一种点杂交膜预处理方法的建立及应用

Pretreatment and Application of Membrane for Dot Hybridization

旨在解决手工点样斑点杂交中样品点密度小、点样体积受限制和点样点不规整等问题,建立了一种新的点杂交膜预处理方法。首先根据预设点样点的大小、排列方式和密度,制作柱状凸起模具;其次将模具固定于杂交膜上,使模具凸起部位与膜紧密接触;再将熔化的石蜡涂布于膜上非模具接触部位,待石蜡凝固后移走模具,获得预处理杂交膜。以荧光染料IRDye800标记的抗体为样品进行直接点样检测,同时提取水稻、小麦和大豆的蛋白进行点杂交实验验证。结果显示,利用上述方法得到非点样部位具有疏水性的预处理NC膜和PVDF膜,其点样点位置、形状和大小固定,点样密度高。不同体积荧光染料标记抗体直接点样得到的荧光图像整齐规范,水稻、小麦和大豆蛋白与水稻看家蛋白抗体Anti-HSP杂交后均能检测到稳定信号。杂交膜经预处理后可最大限度实现点样点位置、大小和形状的统一,并能显著提高上样体积和点样密度。

In order to solve the problems of the low density, the volume limitation, and the irregular shape of sample point in dot hybridization when samples were manually loaded, the hybridization membrane was pretreated with low melting paraffin waxes to fix sample position, size and shape. Firstly, according to the designed size, arrangement and density of sample location on membrane, different columnar salient moulds could be made. After placed a mould on membrane and tightly closed the columnar salient to the membrane, heated waxes was covering on. When waxes become solidification, the mould was removed and pretreated hybridization membrane was obtained. Using pretreated membrane, fluorescent dye IRDye800 labeled protein sample was directly loaded and detected. And finally, the protein samples of rice, wheat, and soybean were extracted, and detected by dot hybridization assay using this membrane. Results showed that pretreated NC and PVDF membrane with hydrophobic area outside of sample point could be obtained by using this method, and the densities, locations, shapes and sizes of sample points could be designed in advance. All fluorescence images of sample points, onto which different volumes of an antibody tagged with a fluorescent dye were loaded directly, showed similar dimensions. Proteins from leaves of rice, wheat and soybean were also detected with Anti-HSP antibody and stable signals could be got. Our results show that when samples were manually loaded on the pretreated membrane, the position, size and shape of sample points can be unified, and the volume and density of samples can significantly improved.