PMC:4307275 / 33524-34698 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4307275","sourcedb":"PMC","sourceid":"4307275","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4307275","text":"The protoplast pellet was suspended in 160 μL of phosphate buffered saline (PBS) (Caisson Laboratories, North Logan, UT, USA) and 40 μL of 5× SDS-PAGE loading buffer (Biosesang, Seongnam, Korea) and heated in boiling water for 10 min. The boiled protoplasts were centrifuged at 4 °C; 5 μL of the supernatant was loaded to each of two 12% acrylamide gels for SDS-PAGE. Following the migration, the proteins in one gel were stained with EZ-Silver Staining Kit for Protein (Biosesang) to visualize the loaded amount and quality, while the proteins in the other gel were transferred to PVDF membrane (Whatman, Kent, UK) using Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). The sGFP chimeric proteins were blotted using rabbit polyclonal antibody of GFP (Abcam, Cambridge, UK) at 1:5000 dilution and secondary anti-rabbit alkaline phosphatase conjugate (Promega, Madison, WI, USA) at 1:5000 dilution. The blots were incubated with Novex® AP Chemiluminescent Substrate (CDP-Star®) (Invitrogen). The developed bands were imaged using a LAS-4000 luminescence detector; the intensity of the bands was quantified using the LAS 4000 software (Fujifilm, Tokyo, Japan).","tracks":[]}