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1) Product Images from "The structure of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR-fold"
Article Title: The structure of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR-fold
Figure Legend Snippet: Mutating the cation-binding residues of SRCR domains abolish function. Through multiple ligand binding assays, we demonstrated the functional importance of calcium binding by the SRCR domains. Mutations affecting site 2 (D1019A) and mutations affecting sites 2 and 3 (D1020A) both abolish function. (A) WT and mutant forms of SRCR8 were incubated with hydroxyapatite beads in a Ca 2+ containing buffer. After extensive washing, bound protein was eluted with EDTA. Eluted fractions were run on a 4 - 20 % SDS-PAGE gel and visualized by Coomassie staining. Only WT SRCR8 bound hydroxyapatite. (B) WT and mutant forms of SRCR8 were flown over a Heparin (HiTrap HP, 1 ml) column in a Ca 2+ -containing buffer. Protein bound to the column was eluted with 0.5 M EDTA. Only WT SRCR8 bound the Heparin-column. Traces: SRCR8 (blue), D1020A (pink), D1019A (red), conductivity (brown). (C) In an ELISA-based setup, a concentration range of the Spy-2 domain of Spy0843 was coated (0.032 – 3.2 μM). WT and mutant SRCR8 domains were added in molar excess (7.1 μM), and binding was detected with a monoclonal anti-SALSA antibody. Binding was only observed for WT SRCR8. (D) Overview of ligand binding studies, + denotes binding, − denotes no binding.
Techniques Used: Binding Assay, Ligand Binding Assay, Functional Assay, Mutagenesis, Incubation, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay
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Article Snippet: Cell pellets from single wells of a 24-well plate of infected cells were washed once with PBS and embedded in molten agarose plugs. .. The plugs were incubated in a solution containing SDS,
Article Title: A Ribosomal Misincorporation of Lys for Arg in Human Triosephosphate Isomerase Expressed in Escherichia coli Gives Rise to Two Protein Populations
Article Snippet: Size Exclusion Chromatography Analysis of HsTIM that had been expressed in E. coli and the P1 and P2 proteins were performed in a Superdex 200 10/300GL analytical column (GE Healthcare) on an Ákta FPLC System (GE Healthcare). .. Generally 300 µl of 20 mM triethanolamine, 0.2 mM
Article Title: Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotogamaritima: Carbon Transfer without Covalent Pyrimidine Activation
Article Snippet: Preparation of Enzyme for Reactions Enzyme was prepared for oxidative half-reactions by being exchanged into 0.1 M