Samples:
1 - Recombinant protein MBP-C terminal region of AHA2, phosphorylation treatment
2 - Recombinant protein MBP-C terminal region of AHA2, subsequently dephosphorylated
3 - Recombinant protein MBP, phosphorylation treatment
4 - Recombinant protein MBP-C terminal region of AHA2, Thr947Ala, phosphorylation treatment 5- Mark: MW markers
1 µg/well of recombinant protein produced in E. coli and purified by affinity chromatography was incubated in the reaction mixture (30 ul). 3 ul of the reaction were mixed with 6 ul Laemli 2X and denatured at 95°C for 5 min. Samples were separated on 10% SDS-PAGE and blotted overnight (ON) to PVDF (Inmobilon®-FL) (pore size of 0.45 µm) using wet transfer. The blot was blocked with 3 % non-fat milk 2h/RT with agitation. The blot was incubated in the primary antibody at a dilution of 1:10 000 for 90 min/RT with agitation 3% non-fat milk TBS 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. The blot was incubated in a matching secondary antibody (anti-rabbit IgG horseradish peroxidase conjugated) diluted to 1: 5000 for 90 min/RT with agitation. The blot was washed as above and developed with the following chemiluminescent detection reagent: AgriseraBright. Exposure time was 2 min.
Courtesy of Cristian Mayordomo, IBMCP, Universidad Politécnica de Valencia, Spain
Titration of the AS22 4789: AHA2 p | ATPase 2 phosphorylated, plasma membrane-type antibody. Ten µg of protein from Amaranth stem mixed membranes (TO), or from beetroot plasma membranes either pre-phosphorylated, i.e. treated with ATP (MP+P), or not (MP* and MP-P) were blotted and probed with the indicated dilutions of antibody. Molecular mass markers (MPM) have, from top to bottom 250, 130, 100, 70 (red) and 55 kDa, respectively.
Courtesy Dr. Luis González, CINVESTAV, Mexico
