Anti-PDLP1 | Plasmodesmata-located protein 1

17 µg/µl of proteins extracted freshly from purified plasmodesmata fraction from Arabidopsis thaliana liquid cell culture were denatured with Laemmli buffer at 50°C for 30 minutes. Separated on 12% SDS-PAGE and blotted to nitrocellulose using semi-dry transfer. Blot blocked with 5% milk for 1 h/RT. Rinsed once with TBS-0.1% Tween and incubated O/N with primary antibody in 5% BSA TBS-0.1% Tween. Rinsed 6 x/ 5 min in TBS-0.1% Tween and incubated with secondary (anti-rabbit IgG horse radish peroxidase) diluted 1 : 10 000/1h. Rinsed 6 x/ 5 min in TBS-0.1% Tween with last wash being TBS only. Incubated with chemiluminescent reagent, according to manufacture's instructions. Exposure time: 60 seconds.

Courtesy of Dr. Emmanuelle Bayer, Laboratory of Membrane Biogenesis, CNRS- Université Bordeaux, France

Each lane contains 20 µg of Arabidopsis thaliana protein. Cotyledons and hypocotyls of seedlings were homogenized and incubated with a detergent solution (sodium deoxycholate) for different amounts of time. After centrifugation proteins were extracted from the pellet with 8 M UTU (6 M Urea and 2 M Thiourea) or precipitated from the supernatant with Acetone.

20 µg/well of total protein extracted freshly from hypocotyls and cotyledons of seedlings.  Exact buffer components were: 100 mM Tris-HCL pH 8, 150 mM NaCl, 10 mM EDTA, 1 mM DTT, 10% Glycerol, 2 mM Na-Deoxycholate, protease inhibitor cocktail. Proteins were denatured with 70 °C 10 min.  Samples were separated in 12% SDS-PAGE and blotted for 1h to nitrocellulose, using: semi-dry transfer. Blot was blocked with Roche Western Blotting Reagent (11921681001) for 1 h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:  5000 with agitation in TBS-T ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed three times for 15 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody diluted to 1: 25 000 in  for 1h/RT with agitation (AS09 602). The blot was washed as above and developed with a following chemiluminescent detection reagent: AS16 ECL-N-10. Exposure time was ~15 minutes.

Courtesy of Dr. Sven Gombos, Universität Hohenheim, Stuttgart, Germany