Journal: Journal of Biological Chemistry

Article Title: Biochemical Properties of CikA, an Unusual Phytochrome-like Histidine Protein Kinase That Resets the Circadian Clock in Synechococcus elongatus PCC 7942

doi: 10.1074/jbc.m213255200

Figure Lengend Snippet: FIG. 4. Chromophore ligation assays with PCB and purified CikA variants. Zinc-dependent fluorescence of purified CikA variant holoproteins with PCB is shown as an inverted image. A, control exper- iment using purified CikA6HIS without or with PCB for chromophore ligation assays. B and C, upper panels, each recombinant protein (2 g) loaded on 8% SDS-PAGE was visualized by Coomassie Brilliant Blue (CBB Stain) to check protein concentrations and mobilities. Lower panels, zinc-induced fluorescence from CikA variants (6 g each) with 2 M PCB was visualized on PVDF membranes (Zn Stain). D, zinc- induced fluorescence assay from recombinant CikA6HIS produced in E. coli (rCikA) with added PCB and CikA6HIS purified from S. elongatus (sCikA) with no added PCB.

Article Snippet: The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immun-BlotTM; BioRad) for 3 h at 45 V. After incubation of the PVDF membranes with 1.3 M zinc acetate, the bilin adducts were visualized by fluorescence with a Molecular Dynamics Storm 860 instrument with a setting of red fluorescence and Photomultiplier tube voltage 1000.

Techniques: Ligation, Purification, Fluorescence, Variant Assay, Control, Recombinant, SDS Page, Staining, Produced

Journal: Journal of Biological Chemistry

Article Title: Biochemical Properties of CikA, an Unusual Phytochrome-like Histidine Protein Kinase That Resets the Circadian Clock in Synechococcus elongatus PCC 7942

doi: 10.1074/jbc.m213255200

Figure Lengend Snippet: FIG. 6. Autophosphorylation activity of partially purified CikA6HIS apoprotein. A, temperature dependence of autophosphoryl- ation activity of 1 g of purified protein in the presence of [-32P]ATP at 0, 4, 15, 25, 37, and 42 °C. Incubation in standard reaction buffer was for 15 min. B, time course of autophosphorylation activity of 1 g of purified protein in the presence of [-32P]ATP after 1, 5, 10, 15, 30, 45, and 60 min at 25 °C. C, concentration dependence of autophosphoryla- tion activity with 1 g of affinity-purified Cik6HIS and 10 min of incu- bation at 25 °C with 0, 1, 10, 50, 100, 250, and 500 nM [-32P]ATP. D, competition experiment with [-32P]ATP and 1, 10, 50, 100, 500, or 1000 nM unlabeled ATP. Other components of the reaction mixture were standard, and the incubation was carried out for 10 min at 25 °C. E, chemical stability of phosphoryl linkage after 1 h of treatment at 37 °C with 2 M NaOH, 0.8 M NH2OH pH 6.8, 1 M HCl, 50 mM Tris-HCl, pH 7.5, and no treatment (lanes 1–5, respectively). Incubation was in standard stock buffer with 1 g of purified protein for 15 min at 25 °C. The gel was electroblotted onto PVDF membranes and exposed to a phosphor- imaging plate for 15 min prior to chemical treatment and re-exposed for 15 min after treatment.

Article Snippet: The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immun-BlotTM; BioRad) for 3 h at 45 V. After incubation of the PVDF membranes with 1.3 M zinc acetate, the bilin adducts were visualized by fluorescence with a Molecular Dynamics Storm 860 instrument with a setting of red fluorescence and Photomultiplier tube voltage 1000.

Techniques: Activity Assay, Purification, Incubation, Concentration Assay, Affinity Purification, Imaging