Anti-RbcS I Ribulose bisphosphate carboxylase small subunit (cyanobacterial)

samples:
1 - 29 µg of Synechocystis sp. PCC6803 whole cell extract.
2 - 14 µg of Cyanobium sp. PCC 7001 whole cell extract.
3 - 19 µg of Synechococcus sp. PCC 7002 whole cell extract.
4 - 14 µg of Synechococcus elongatus PCC 7942 whole cell extract.
5 - 14 ug of Synechococcus sp. PCC 11901 whole cell extract.
6 - Halothiobacillus neapolitanus whole cell extract.
7 - 6.8 µg of purified Rubisco from spinach leaves.

Note: Protein load/well of respective extract is not the same, therefore differences in intensity of visualized RbcS bands.

About 20 µg/well of total protein extracted freshly from respective cyanobacteria, spinach or Halothiobacillus neapolitanus were extracted with 50 mM Tris-HCl, pH 8.0, 20 mM MgCl2, 20 mM NaHCO3, 0.2 mM EDTA, and denatured with exact buffer components at 95 °C for 10 min. Samples were separated in the cold on 17% SDS-PAGE and blotted for 1 h to PVDF or (pore size of 0.2 µm), using wet transfer in the cold. Blot was blocked with 5 % milk for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5 000 for ON/4°C 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. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 5 000 in TBS for 1 h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent. Exposure time was 1 minute.

Courtesy of Dr. Yu Chen, Department of Biochemistry, Cell and Systems Biology Institute of Systems, Molecular and Integrative Biology University of Liverpool, United Kingdom