mNeonGreen | Fluorescent Protein

Samples: 
1 - Nicotiana benthamiana whole leaf extract expressing mNeonGreen-ATG8
2 – Nicotiana benthamiana whole leaf extract expressing YFP-ATG8
3 – Nicotiana benthamiana whole leaf extract expressing mCherry-ATG8

MW markers: (Biorad)Precision Plus Protein Dual Color Standards, 500 µl #1610374

50 µg of total protein extracted freshly from Nicotiana benthamiana leaves with exaction buffer (50mM Tris-HCl pH7.5, 150mM NaCl, 10%(v/v) glycerol, 0.5mM EDTA and 0.5% NP40) denatured with SDS-sample buffer at 95°C for 5 min, proteins were separated on 10 % SDS-PAGE and blotted 0.5h to PVDF membrane using semi-dry transfer. Blots were blocked with 5 % milk in PBS-T for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 3 000 for ON/4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in PBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG (H&L), horse radish peroxidase conjugated, AS09 602) diluted to 1: 5 000 in for 1h/RT with agitation. The blot was washed as above, developed for 5 mins with Agrisera ECL Bright and detected with film.

Courtesy of Dr. Jiaqi Sun, McGill University, Canada

Samples:

1- PageRuler™ Prestained Protein Ladder (Thermo Fisher)
2- 20 µg Arabidopsis thaliana wildtype microsomal membrane preparation
3- 20 µg Arabidopsis thaliana expressing VHA-a1-GSL-NeonGreen microsomal membrane preparation
4- 20 µg Arabidopsis thaliana expressing VHA-a3-GSL-NeonGreen microsomal membrane preparation

20 µg of microsomal membrane proteins were prepared from Arabidopsis thaliana 4-week -old rosette leaves with a buffer containing 350mM sucrose, 70mM Tris-HCl pH8, 10% glycerol, 3mM Na2EDTA, 0.15% BSA, 1.5% PVP-40, 4mM DTT, 1x Roche complete^TM protease Inhibitor and denatured at 95ºC for 5 minutes. Proteins were separated on 10 % SDS-PAGE and blotted for 1 h to PVDF membrane (pore size 0.2 µm) using wet transfer. The blot was blocked with 5% milk/TBS-T (0.05%) for 1 h at RT with agitation and then incubated in the primary antibody at a dilution 1: 1000 in 5% milk/TBS-T (0.05%) for 1h at RT with agitation. The antibody solution was decanted, and the blot was briefly rinsed, then washed three times for 10 minutes in TBS-T (0.05%) with agitation. The blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG, HRP conjugated, AS09 602) at a dilution of 1: 25 000 in 5 % milk/TBS-T (0.05%) for 1hr at RT with agitation. The blot was washed as above, developed for 1 min with AgriseraECLBright substrate and imaged with a cooled CCD camera system (Intas ADVANCED Fluoreszenz u. ECL Imager). Exposure time was 10s.

Courtesy of Upendo Lupanga, Centre for Organismal Studies, University of Heidelberg, Germany

• dCas9-P2A-DHFR(TSc3)-T2A-mNeonGreen [Cloning vector pBM020]
• Cas9-P2A-DHFR(TSc3)-T2A-mNeonGreen [Cloning vector pBM006]
• mNeonGreen4-tDeg [Cloning vector pminiCMV-mNeonGreen4-tDeg]
• ER-localized mNEONGREEN [Binary vector pKT-NM-erNEON]
• mNeonGreen-3xFLAG [Cloning vector pLM160-mNeonGreen]
• mNeonGreen-ty1 [Cloning vector pSAG1-mNeonGreen_TUB1-dTomato]
• mNeonGreen, partial [Binary vector pRATIO2131]