V-ATPase, a3 | vacuolar H+-ATPase subunit a isoform 3

30 µg of microsomal membranes were extracted freshly from 5-day-old Arabidopsis thaliana seedlings grown in liquid culture with a buffer containing 350mM sucrose, 70mM Tris-HCl pH8, 10% glycerol, 3mM Na2EDTA, 0.15% BSA, 1.5% PVP-40, 4mM DTT, 1x Roche completeTM Protease Inhibitor and denatured with 1x Lämmli buffer at 50°C for 10 min. Proteins were separated on 7,5 % SDS-PAGE and blotted 1h to PVDF (pore size of 0.2 µm) using wet transfer. Blot was blocked with 3% milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 in 2%BSA in TBS-T (0.05%) for ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly with TBS-T, then washed twice for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:25 000 in 3% milk in TBS-T for 2h/RT with agitation. The blot was washed as above and developed for 2 min with chemiluminescent detection reagent. Exposure time was 100 seconds.

expected band sizes: 1 - marker as indicated in the picture 2 - endogenous VHA-a3 at ~93kDa 3 - endogenous VHA-a3 at ~93kDa 4 - no band since it is the mutant (It also tells us that the AB is not recognizing VHA-a2 which is very similar to VHA-a3) 5 - no band since it is the mutant (It also tells us that the AB is not recognizing VHA-a2 which is very similar to VHA-a3) 6 - endogenous VHA-a3 at ~93kDa (It also tells us that the AB is not recognizing VHA-a1(-GFP) which is similar to VHA-a3) 7 - endogenous VHA-a3 at ~93kDa and VHA-a3-GFP at ~120kDa

Courtesy of Upendo Lupanga, Centre of Organismal Studies, University of Heidelberg, Germany