The tissue was ground in liquid nitrogen, resuspended in cold lysis buffer (20 mM HEPES pH 7.2, 2 mM DTT, 250 mM sucrose, 1 mM MgCl2, 2.5 U/mL benzonase), and incubated on ice (15–30 min). Protein concentrations were determined by a Quick StartTM Bradford Protein Assay and diluted samples were flash-frozen in liquid nitrogen and stored at −80 °C until further use.
The sample was diluted to 2 mg/mL, and 50 μL was absorbed. The probe was balanced in desiccant to room temperature, and 100 μL DMSO was added to prepare a 0.1 mm storage solution. 1 μL of ActivX® Serine Hydrolase Probes (Thermo, 88318) was added to each sample at a final concentration of 2 μm/μL. After mixing, the samples were incubated for 1 h at room temperature under the light. The reaction was terminated by boiling 10 μL 6X SDS–PAGE protein loading buffer for 5 min. The reaction was electrophoresed with 12% SDS–PAGE. Gels were scanned using Cy3 and Cy5 multichannel settings (605/50 and 695/55, filters respectively) and stained with Coomassie after scanning. Fluorescence intensity was analyzed by Image J. The SDS–PAGE with fluorescence coloring was placed in Coomassie brilliant blue glue dye solution and stained on a shaker for 2 h. Then, the molecules with fluorescence coloring and Coomassie brilliant blue staining were decolorized and scanned, and the different bands were cut out for identification.
The sample was diluted to 2 mg/mL, and 50 μL was absorbed. The probe was balanced in desiccant to room temperature, and 100 μL DMSO was added to prepare a 0.1 mm storage solution. 1 μL of ActivX® Serine Hydrolase Probes (Thermo, 88318) was added to each sample at a final concentration of 2 μm/μL. After mixing, the samples were incubated for 1 h at room temperature under the light. The reaction was terminated by boiling 10 μL 6X SDS–PAGE protein loading buffer for 5 min. The reaction was electrophoresed with 12% SDS–PAGE. Gels were scanned using Cy3 and Cy5 multichannel settings (605/50 and 695/55, filters respectively) and stained with Coomassie after scanning. Fluorescence intensity was analyzed by Image J. The SDS–PAGE with fluorescence coloring was placed in Coomassie brilliant blue glue dye solution and stained on a shaker for 2 h. Then, the molecules with fluorescence coloring and Coomassie brilliant blue staining were decolorized and scanned, and the different bands were cut out for identification.
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