Journal: FEBS Open Bio

Article Title: Characterization of an intertidal zone metagenome oligoribonuclease and the role of the intermolecular disulfide bond for homodimer formation and nuclease activity

doi: 10.1002/2211-5463.12720

Figure Lengend Snippet: Effect of Mn 2+ (A) and Mg 2+ (B), pH (C), and NaCl (D) on the nuclease activity of MG Orn. The effect of the metal ions and pH on the enzyme activity has been determined using the time‐resolved p NP ‐ TMP activity assay at 25 °C as described in with 1.1 μg Orn and varying amounts of Mn 2+ and Mg 2+ as well as 1.5 μg MG Orn and pH values from 6.5 to 9 using MES ( pH 6.5), HEPES ( pH 7–7.5), and Tris ( pH 8–9) as indicated in A–C. The rate of hydrolysis of p NP ‐ TMP at the varying pH values was calculated according to Hamdan et al . . Error bars indicate the standard deviation of the measurements. The effect of NaCl in a range of 0–2 m is shown in (D) and has been tested at 25 °C with the gel‐based nuclease activity assay with 0.05 μ m 7mer RNA substrate and 0.8 μg MG Orn in 50 m m Tris‐ HC l pH 8.0, 0.2 mg·mL −1 BSA , 2% glycerol and 1 m m MnCl 2 for 15 min. Samples were analyzed on 20% denaturing PAA gels (8 × 8 cm). Reaction buffer was used as negative control (Neg) instead of protein solution.

Article Snippet: The reactions contained 50 m m HEPES pH 7.5 (at 25 °C), 72 m m NaCl, SYPRO ® Orange (Merck KGaA) in a final dilution of 6× and 4 μg of protein.

Techniques: Activity Assay, Standard Deviation, Negative Control

Journal:

Article Title: Regulation of InsP 3 receptor activity by neuronal Ca 2+ -binding proteins

doi: 10.1038/sj.emboj.7600037

Figure Lengend Snippet: CaBP1 binds directly to the InsP3R. (A) SDS–PAGE gel, stained with SYPRO Orange™, showing the interaction between either 100 μg pGST, GST 1–604, GST 1–225, GST 226–604 or 100 μg SCaBP1 in a pull-down assay in the presence of 200 μM free Ca2+. In lanes 2 and 3, SCaBP1 was retained by GST 1–604 and GST 1–225, respectively. (Bi) Map showing positions of synthetic peptides (A–F) used for binding experiments relative to the NH2-terminal 159 aa region of the InsP3R. Peptide A (S16-N48), peptide B (P49-N81), peptide C (Y66-K91), peptide D (D97-L123), peptide E (E106-S128) and peptide F (I121-L151). White bars indicating the Ca2+-independent CaM binding sites on the InsP3R1 (Sienaert et al, 2002). Grey bar (B) showing a positive interaction of the peptide with SCaBP1. (Bii) Nondenaturating gel (15%) of 3.5 μM SCaBP1 in the presence of each of the InsP3R1 peptides (A–F) (35 μM) in 50 mM Tris buffer (pH 7.4) containing either 200 μM free Ca2+ or 1 mM EGTA stained with SYPRO Orange™. In lane 1, SCaBP1 alone was used as a control. SCaBP1 bound to the peptide diminishes the intensity of the SCaBP1 band. (C) SDS–PAGE gel showing the interaction of SCaBP1 with increasing amounts of peptide B (P49-N81) in the presence of 200 μM Ca2+ or 1 mM EGTA stained with SYPRO Orange™. (Lower panel) Densitometric analysis of the SCaBP1 bands by ImageQuant 5.2 of the interaction between SCaBP1 and peptide B (P49-N81) in the presence of 200 μM Ca2+ (triangles) or 1 mM EGTA (squares). The vertical axis denotes the intensity of the SCaBP1 band. (D) co-IP of InsP3R3 from COS-7 cells transfected with either SCaBP1 or LCaBP-YFP. YFP-CaBP1 was immunoprecipitated with anti-GFP antibody. InsP3R3 immunoreactivity was detected using an anti-InsP3R3 monoclonal antibody and subsequent ECL. Transfected cDNA is indicated above the blot. (E) co-IP of InsP3R3 with SCaBP1-YFP was performed in the absence (lane 3) or presence of 1 mM EGTA (lane 4). Control IPs from YFP-transfected cells and nontransfected cells were also performed (lanes 1 and 2). (F) co-IP of CaBP with InsP3R1 from the brain. The antibody used for the IP is indicated below the blot and the antibody used for the Western blot (WB) is indicated above the blot. C indicates IP with a non-specific antibody. (Fi) IP of InsP3R1 subsequently detected by Western blot with anti-InsP3R1 antibody. (Fii) Parallel IP using an anti-InsP3R1 antibody and Western blot using an anti-CaBP antibody.

Article Snippet: Analysis of the eluted proteins was performed on NuPAGE ® gels, 4–12% Bis–Tris in MES–SDS buffer (Invitrogen Life Technologies) and detected by Sypro™ Orange staining (Amersham Pharmacia Biotech).

Techniques: SDS Page, Staining, Pull Down Assay, Binding Assay, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot