A modified protein persulfidation detection method

Abstract

As a gastransmitter, hydrogen sulfide (H2S) plays an important role in regulating plant growth and stress resistance. The proposed functional mechanism of H2S is persulfidation, a kind of posttranslational modification on free cysteine residues of proteins, which is also called S-sulfhydration previously. At present, biotin switch assay is an effective method for protein persulfidation level detection, while the protein sample need be precipitated and resuspended repeatedly according to the current protocol. Here, we reported a modified method which adapted from biotin switch assay and reduced the repetitive steps mentioned above. The total protein of plant or purified recombination protein from E. coli was directly loaded on the activated NC membrane. The free sulfhydryl of cysteine residues of protein on NC membrane could be blocked with MMTS reagent, while the persulfidation modified cysteine residues would be labeled by biotin-HPDP. After that, the persulfidation level of proteins was detected by immunoblotting with anti-biotin antibodies. This method was also applicable to the plant proteins, which were transferred to NC membrane after separated by native PAGE. This method reduces the repeated steps of precipitation and resuspension of protein samples, and by this it could reduce the loss and content error of protein. Besides, it is more sensitive and convenient than the previous biotin switch assay method.