4.5. Analysis of sGFP Expression in Protoplasts
For the expression analysis, protoplasts (approximately 1 × 106) were centrifuged, the supernatant was removed, and the pellet was immediately frozen in LN2 and stored at −80 °C until RNA and protein extraction.
Total RNA was extracted from rice protoplasts using the RNeasy® Plant mini kit (Qiagen, Hilden, Germany) and the 1st strand cDNA was synthesized using a mRNA selective PCR kit (AMV) ver. 1.1 (Takara, Shiga, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using a Thunderbird™ SYBR® qPCR mix (Toyobo, Osaka, Japan); the SYBR-fluorescence signals were detected and quantified using a CFX96™ Real-Time PCR system (Bio-Rad, Foster City, CA, USA). The quantified SYBR-fluorescence of sGFP transcript was analyzed using CFX Manager™ ver. 2.1 (Bio-Rad). A rice ubiquitin gene (AK061988) was used as a reference gene to normalize the expression data for each sGFP transcript.
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).