V-ATPase, c | Vacuolar H+-ATPase, subunit c (16 kDa)

Following samples were analyzed: MW markers (1), Arabidopsis thaliana tonoplast (2), Arabidopsis thaliana plasma membrane (3), Arabidopsis thaliana microsomes (4), Arabidopsis thaliana total protein (5), Mesembryanthemum crystallinum tonoplast (6), Mesembryanthemum crystallinum plasma membrane (7), Mesembryanthemum crystallinum microsomes (8), Nicotiana tabacum microsomes (9), Brassica napus microsomes (10). 15 µg of the indicated protein, extracted according to Vera-Estrella et al. (2012) was separated on 12% SDS-PAGE and blotted 1.15h to PVDF using tank transfer. Blots were blocked with for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 O/N at RT with agitation. The antibody solution was decanted and the blot was washed 3X for 15 min min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602 ) diluted to 1:50 000 in for 2h at RT with agitation. The blot was washed as above and developed using chemiluminescent substrated and recorder using the LiCOR c-DIGIT personal imager.

Courtesy Dr. Bronwyn Barkla. UNAM, Mexico

Subunit c is one of most hydrophobic proteins (can be dissolved in organic solvent such as a mixture of chloroform/methanol solution). It is prone to aggregation even in the presence of SDS. Therefore, before loading on the gel membrane fractions should be incubated in buffer containing 2 % SDS at 60° or 70°C for 10 min or at 25°C for 30 min.