GFP | Green Fluorescence Protein, monoclonal (clone 2G4:F2)

Samples

20 μg/well of total protein extracted freshly from siliques (stade 20dap, 50mg of powder) and imbibed seeds (50 mg starting material). Exact buffer components were Cell Culture Lysis 1X Reagent (Promega - E153A + inhibitor protease cocktail) and denatured with Laemmli buffer (375 mM Tris.HCl, 9% SDS, 50% Glycerol, 0.03% Bromophenol blue) at 95 °C/5 min. Samples were separated on 12 % SDS-PAGE and blotted for 1 h to nitrocellulose (pore size of 0,45 µm), using wet transfer. Blot was blocked with 5 % milk for 2h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 in 5 % milk TBS-T ON/4°C with agitation. The antibody solution was decanted, and the blot was washed 4 times for 5 min in TBS-T at RT with agitation. Blot was incubated with secondary antibody (AS09 627-trial, Rabbit anti-mouse IgG, HRP conjugated, Agrisera) diluted to 1: 10 000 in 5 % milk for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent AS16 ECL-S-10, AgriseraSuperBright. Exposure time was 20 seconds.

Courtesy of Dr. Victoria Gomez Roldan,French National Centre for Scientific Research, France

Total proteins were isolated from 4 leaves (5-week-old plants) of Arabidopsis thaliana were ground in 400 μL homogenization buffer or from 15 seedlings (7-day-old) ground in 100 μL homogenization buffer as described by LaMontagne et al (2016). Plant lines were Arabidopsis thaliana Col-0 (ecotype, wild-type) and an overexpressing GFP-tagged ABCG-protein (GFP-ABCG ox). 15 μl of total proteins were denatured at 37°C for 5 min, separated on 8% SDS-PAGE and transferred for 70 min at 55V using a tank transfer system to nitrocellulose membrane (0.45 μm). Blots were blocked with PBS+0.1% Tween 20 (PBS-T)+5 % milk at room temperature (RT) with agitation for 2 hours. Monoclonal primary anti-GFP-antibody (AS21 4696, Agrisera; clone 2G4:F2) was diluted to 1:30,000 and incubated with membrane portion overnight at 4°C with agitation in PBS-T+5 % milk. The primary antibody solution was decanted, and blots were washed 4 times (6 minutes each) in PBS-T at RT with agitation. Blot was incubated with secondary rabbit anti-mouse IgG (H&L) HRP conjugated (AS09 627-trial, Agrisera) diluted to 1: 25,000 for 2 hours, washed as described above (4 times with PBS-T at RT), and developed with chemiluminescent detection reagent ECL Bright (AS16 ECL-N, Agrisera) according to manufacture's recommendations Exposure time was 1 second (1s) on X-ray films. Ponceau Stain of the total proteins and anti-MPK6 detection served as loading control.

Courtesy of Nga Nguyen and Dr. Antje Heese at the University of Missouri-Columbia, Div. of Biochemistry, Interdisciplinary Plant Group (IPG), Columbia MO (USA).

Reference: LaMontagne ED, Collins CA, Peck SC, Heese A. Isolation of Microsomal Membrane Proteins from Arabidopsis thaliana. Curr Protoc Plant Biol. 2016 May;1(1):217-234. doi: 10.1002/cppb.20020. PMID: 31725992.