Anti-RA | Rubisco activase

Courtesy of Dr. Katia Wostrikoff, CNRS/Sorbonne, France



Samples from left to right:
1 – MW marker
2 – 10µg of Arabidopsis thaliana whole leaf extract
3 – 10µg of Hordeum vulgare whole leaf extract
4 – 10µg Chlamydomonas reinhardtii whole cell extract

10 µg/well of respective cell extract was denatured with Invitrogen LDS sample buffer (4X)  at 70°C/5 min.  Samples were separated on Invitrogen NuPage Bis-Tris 4-12% SDS-PAGE 45min and blotted for 1 h to Invitrogen PVDF (pore size of  0.45  um), using: wet transfer. Blot was blocked with 5% milk in TBS-T for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 for 1h/RT with TBS-T Blocking. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 10 min and 2 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG ALP conjugated, AS09 607 lot 2105 ) diluted to 1: 5 000 in TBS-T Blocking for 0,5h/RT with agitation. The blot was washed as above and developed with AS19 BCIP-NBT lot 03068231 for 0.5-3min. Image was captured after 2h.

RA - Ribulose bisphosphate carboxylase/oxygenase activase is an enzyme localized to chloroplasts which activates Rubisco by promoting ATP-dependent conformational changes. Alternative name: RuBisCo activase RCA.