CNX1/2 | CALNEXIN HOMOLOG 1/2

CNX1 60.5 kD, processing aa 1-20, mature peptide 58.1 kD

CNX2 60.5/61.4 kD, processing aa 1-25, mature peptides 57.6/58.6 kD

Total protein from Col-0 (wild-type) Arabidopsis thaliana were extracted with 50mM HEPES-KOH buffer containing 250 mM sucrose, 5% glycerol, 50 mM NaPP, 1 mM NaMo, 25 mM NaF, 10mM EDTA, 0.5% PVP, 3mM DTT, 1mM PMSF, 10uM Leupeptin & 10nM Calyculin, and then fractionated by ultracentrifugation at 100,000 x gravity for 30 min at 4°C into soluble (S100) and microsomal (P100) proteins as described in LaMontagne et al. (2016). Isolation of Microsomal Membrane Proteins from Arabidopsis thaliana. Current Protocols in Plant Biology 1:1-18. doi: 10.1002/cppb.20020. 30 µg proteins of total, S100 and P100 fractions were denatured at 37°C for 5 min, separated on a 7.5 % SDS-PAGE and blotted 1h to nitrocellulose using tank transfer. Blots were blocked with 1x PBS (from Fisher Scientific BP665-1) + 0.1 %Tween 20 (PBS-T) + 5% milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2500 overnight at 4°C with agitation in 1x PBS-T + 5% milk. The antibody solution was decanted, and the blot was rinsed briefly once, then washed four times for 7 min in 1x PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in 1x PBS-T + 5% milk for 2 hrs at RT with agitation. The blot was washed as above and developed for 4 min with Amersham ECL (RPN2106). Exposure time was 30 seconds and 2 min

Courtesy of Erica LaMontagne & Dr. Antje Heese (Division of Biochemistry, Interdisciplinary Plant Group (IPG) - University of Missouri; Columbia, MO, USA)

AS09 614 | Anti-BiP | lumenal-binding protein, rabbit antibody
AS07 266 | Anti-SMT1 | Sterol methyltransferase 1, rabbit antibody
AS13 2665 | Anti-KDEL | endoplasmic reticulum retention signal (50 µg) , rabbit antibody
AS10 683 | Anti-HDEL | endoplasmic reticulum retention signal (100 µg), mouse antibody