Anti-CALS12/PMR4 | Callose synthase 12

1: Col-0 – Total protein extract
2: pmr4-1/cals12 – Total protein extract
3: Col-0 – soluble protein fraction (S100)
4: pmr4-1/cals12 – soluble protein fraction (S100)
5: Col-0 – microsomal protein fraction (P100/M)
6: pmr4-1/cals12 – microsomal protein fraction (P100/M)

Total proteins were isolated from 60 Arabidopsis thaliana seedlings (10-day-old) of Col-0 (wild- type; lanes 1, 3, 5) and pmr4-1/cals12 null mutant [ref 1] (lanes 2, 4, 6). Using differential centrifugation, the total protein fractions (lanes 1, 2) were separated into soluble proteins (S100; lanes 3, 4) and microsomal proteins (M/P100; lanes 5, 6) as previously published by our lab [ref 2]. Proteins were denatured at 65°C for 5 min. 30 μg of proteins were separated on an 8 % SDS-PAGE and transferred for 70 min at 55V using a tank transfer system to nitrocellulose membrane. Blots were blocked with 1x PBS + 0.1 % Tween 20 (PBS-T) + 5% milk for 1 h at room temperature (RT) with agitation. To test primary α-CalS12/PMR4 by Agrisera (AS21 4567), different dilutions of the antibody were used as indicated in the figure. Membrane portions probed with α-CNX1/2 (AS12 2365, membrane ER) and α-MPK6 [ref 1] served as loading controls. Blots were incubated with the primary antibodies overnight at 4°C with agitation in 1x PBS-T + 5% milk. The primary antibody solutions were decanted, and the blots were washed 4 times (6-8 minutes each) in 1x PBS-T at RT with agitation prior to incubation with secondary antibody Goat anti Rabbit IgG (H&L) –HRP conjugated (AS09 602-trial) diluted to 1 : 20,000 in 1x PBS-T + 5% milk for 2 h at RT with agitation. The blots were washed as above and developed for 4 min with chemiluminescent detection reagent ECL Bright (AS16 ECL-N). Exposure time to X-ray films was indicated in figure.

Courtesy of Kelly Mason and Antje Heese; University of Missouri, Div. Biochemistry, IPG (USA)

References
[1] Mason K, Ekanayake G, Heese A (2020). Staining and automated image quantification of callose in Arabidopsis cotyledons and leaves. Methods Cell Biology: Plant Cell Biology 160:181-199.
[2] LaMontagne, E.D., Collins, C.A., Peck, S.C. and Heese, A. 2016. Isolation of microsomal membrane proteins from Arabidopsis thaliana. Curr. Protoc. Plant Biol. 1:217-234. doi: 10.1002/cppb.20020