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 designedin 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 andstablesignals 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.
