pi 4 5 p 2 biotin  (Biacore)

Journal: iScience

Article Title: Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function

doi: 10.1016/j.isci.2023.108151

Figure Lengend Snippet: Biophysical analysis of the interactions of DIRAS3 NTE peptides with PIP 3 and PIP 2 (A–D) Representative SPR experiment for binding analysis on a PIP 3/2 diC 8 lipid sensor (biotin-tethered): Peptide solution (2x or 3x dilution in buffer: 20 mM Tris, pH7.5, 150 mM NaCl, 0.02% Tween 20) of indicated concentration of (A) NTE 2-11 , (B) NTE 12-29 , and (C and D) NTE wt at the concentrations indicated were injected onto PI-biotin (reference surface), PI(3,4,5)P 3 -biotin or PI(4,5)P 2 -biotin (∼200 RU, captured by streptavidin immobilized on a Biacore sensor chip surface). SPR sensorgrams (shown in black) were fitted to a two-state binding model (in red), and the derived kinetic rate constants for the binding are listed. See also Figures S3 A and S3B. (E–G) SPR measurement of NTE wt binding to surface tethered liposomes (∼1500 RU for PIP 2 liposome, ∼1700 RU for PIP 3 liposome, and ∼1100 RU for control liposome). The NTE wt bound sensorgram were fitted to a 1:1 binding model (in black) to obtain binding constants ( K D = k d / k a ). (H–K) ITC analysis of the binding reaction. (H) Blank-subtracted ITC data for the titration of PIP 3 diC 8 (500 μM) onto NTE wt peptide (78 μM) showing the thermogram (upper panel) and binding isotherm (lower panel) of integrated data point (black circles) fitted to a multiple-site model (black line, best fit model) using NanoAnalyze Data Analysis software to obtain thermodynamic parameters. (I–J) Blank-subtracted ITC data for the titration of PIP 3 diC 4 or PIP 3 diC 8 (500 μM) onto NTE wt or NTE scr peptide (78 μM). No binding enthalpy can be detected, indicated by scattered data points around a fitted straight line. (K) Binding signatures plotted for thermodynamic parameters of the PIP 3 diC 8 –NTE wt interaction. The average values (from two independent experiments, standard deviation in parentheses) of standard free energy (ΔG), binding enthalpy (ΔH), and entropy contribution (-TΔS) are shown for the binding of PIP 3 to site 1 and 2 on NTE wt . The stoichiometry of binding (n), and apparent equilibrium-dissociation constant ( K D ) are also listed.

Article Snippet: Biophysical analysis of the interactions of DIRAS3 NTE peptides with PIP 3 and PIP 2 (A–D) Representative SPR experiment for binding analysis on a PIP 3/2 diC 8 lipid sensor (biotin-tethered): Peptide solution (2x or 3x dilution in buffer: 20 mM Tris, pH7.5, 150 mM NaCl, 0.02% Tween 20) of indicated concentration of (A) NTE 2-11 , (B) NTE 12-29 , and (C and D) NTE wt at the concentrations indicated were injected onto PI-biotin (reference surface), PI(3,4,5)P 3 -biotin or PI(4,5)P 2 -biotin (∼200 RU, captured by streptavidin immobilized on a Biacore sensor chip surface).

Techniques: Binding Assay, Concentration Assay, Injection, Derivative Assay, Liposomes, Titration, Software, Standard Deviation

Journal: Biochemical Journal

Article Title: Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain

doi: 10.1042/BCJ20160791

Figure Lengend Snippet: ( A ) Schematic representation of SPR experiments with the immobilised lipid headgroup as the ligand and the PH domain as the analyte. ( B ) SPR measurements of the kindlin-3 PH domain WT and mutants with immobilised PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 , respectively. On the left in each case, we show the background-subtracted raw sensogram data for the WT protein, while on the right we show binding saturation curves for WT, K363A K367A and IPRR insertion mutants. The highest concentration used is given on each set of sensogram curves; subsequent binding profiles derive from a series of twofold dilutions.

Article Snippet: PtdIns(4,5)P 2 (C-45B6a) and PtdIns(3,4,5)P 3 (C-39B6a) were purchased from Echelon Biosciences.

Techniques: Binding Assay, Concentration Assay