PGRL1 | PGR5-like protein 1A (chloroplastic)

Whole leaf protein (corresponding to 3 mg fresh weight) from the Arabidopsis thaliana lines Columbia-0 (Col-0), PGR5 knock out (pgr5-1), line-2 (line-2) and PGRL1A+B knock out (pgrl1ab) was extracted and denatured with 2x Tricine sample buffer (100mM Tris/HCl, 24% (w/v) glycerol, 8% SDS and 15mM DTT) by heating for 5 min at 70°C. The samples were separated on a 10% Tris Tricine gel and blotted overnight on PVDF-membrane using capillary blotting. The membrane was blocked with 5% milk in Tris-Buffered saline and 0.1% Tween (TBS-T (50mM Tris, 150mM NaCl, 0.1% Tween, pH 7.5)) for 1h at room temperature with agitation and subsequently incubated in the primary antibody at a dilution of 1: 5000 overnight at 4°C with agitation in 1% milk in TBS-T. The antibody solution was decanted and the blot was rinsed briefly, then washed 3 times for 10 min in TBS-T at RT with agitation. The membrane was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602) diluted to 1:10 000 in 1% milk in TBS-T for 1h at RT with agitation. The blot was washed 3 times for 10 min in TBS-T, once for 5 min in TBS (50mM Tris, 150mM NaCl, pH 7.5) and developed for 5 min with chemiluminescent detection reagent  according to manufacture's recommendations. Exposure time was 5 seconds.

Courtesy of Jan-Ferdinand Penzler from Ludwig-Maximilians-University Munich (LMU), Dario Leister Lab, Germany

Experimental conditions for detection in extracts of Picea abies and Pinus sylvestris:

25 µg/well of total protein extracted  from needles of Picea abies and Pinus sylvestris.   Exact buffer components were: 10% trichloroacetic acid (TCA)/ acetone buffer. Then followed by a methanol buffer (80% methanol with 0.1 M ammonium acetate) wash and an 80% acetone wash step. Subsequently, total of 1.8 ml mixture of 1:1 phenol (pH 8.0)/SDS buffer (30% sucrose, 2% SDS, 0.1M Tris-HCl, pH 8.0, 5% 2-mercaptoethanol) were added to the tube to extract proteins from dry pellets. After centrifuging at 16,000 g, 4 °C for 3 min, 0.4 ml phenol phase extraction were mixed with 1.6 ml methanol buffer and then centrifuged at 16,000 g, 4 °C for 3 min. The pellets were washed twice with methanol and 80% acetone. Final protein pellets were dissolved in 0.2 ml Laemmli sample buffer,  and denatured at 100 °C for 10 min.  Samples were separated in the cold on 12 % SDS-PAGE  and blotted for 1 h to PVDF membrane, using: wet transfer in the cold. Blot was blocked with 5 % milk for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 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 TBS-T at RT with agitation. Blot was incubated in  matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 10 000 in  for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent:. Exposure time was  1-5 minutes. Yang et al. 2020.