PDLP1 | Plasmodesmata-located protein 1

17 ug/ul 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 1h/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 8M UTU (6M Urea and 2M 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 1h/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