GAPC1/2 | Glyceraldehyde-3-phosphate dehydrogenase

Samples:
1 – Synechocystis sp. PCC 6803 wild type, 10 µg total protein extract
2 – Synechocystis cp12 mutant, 10 µg total protein extract
3 – Synechocystis gap1 mutant, 10 µg total protein extract
4 – Arabidopsis thaliana wildtype, 10 µg whole leaf extract

10 µg of total protein extracted freshly from Synechocystis sp. PCC 6803 with 1x PBS. Buffer components: 137 mM NaCl, 10 mM Na2HPO4*2H2O, 2 mM KH2PO4, 2,7 mM KCl, pH 7.4. 10 µg of whole leaf extract extracted from Arabidopsis thaliana. Extraction buffer components: 50 mM HEPES, 10 mM NaCl, 5mM MgCL₂*6H₂O, 100 mM Sorbitol, pH 7.6. Denatured at 95°C for 5 minutes with 3x Laemmli buffer, components: 150mM Tris-HCl (pH 6.8), 300 mM DTT, 6% SDS, 0.3% bromophenol blue, 30% glycerol. Samples were separated on 12% SDS-PAGE and blotted 90 minutes to PVDF membrane, using semi-dry transfer. Blot was blocked with 5% milk in 1xTBS for 1h at RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 with agitation in 1xTBS overnight at 4°C. The antibody solution was decanted, and the blot was rinsed twice, then washed 4 times for 10 minutes in 1xTBS at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in 5% milk in 1xTBS for 2h/RT with agitation. The blot was washed as above and developed with AgriseraECLBright chemiluminescent solution. Exposure time was 10 seconds.

Courtesy of Dr. Stefan Lucius, Universität Rostock, Germany

40 μg total proteins from Arabidopsis wt, and GAPC1-6xFlag, gapc1-1, gapac1-1/gapc2-1, and gapc1-1/gapc2-2 mutants, extracted and denatured as described by Larkin (2007), were separated on  12% SDS-PAGE  and blotted 1h to PVDF using tank transfer. Blots were blocked with TBS-T buffer with 5% skimmed milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 for overnight at 4°C  with agitation in TBS-T with 2% skimmed milk. 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. Blot was incubated in secondary antibody (Goat anti-rabbit IgG horse radish peroxidase conjugated, from ZSGB-BIO, China) diluted to 1:0000 in  for 1h at RT with agitation. The blot was washed as above and developed for 1 min with EasySee Western Blot Kit (TRANS^TM, China). Exposure time was  30 seconds.

Courtesy of Dr. Songhu Wang, Chengdu Institute of Biology, Chinese Academy of Sciences, China