PIP strip membranes (Life Technologies) were blocked in blocking buffer (3% fat-free bovine serum albumin [Sigma] in TBS [20 mM Tris-HCl, pH 7.5, 500 mM NaCl]) for 1 h at room temperature with mild agitation. Blocked PIP strips were then incubated in 0.4 µM MBP-Stv1NT-FLAG in blocking buffer for 1 h. Bound protein was detected by incubation with mouse monoclonal anti-FLAG M2 antibody (Sigma) followed by horseradish peroxide–conjugated anti-mouse antibody (Bio-Rad). All incubations were for 1 h at room temperature and were followed by three TBS washes for 10 min. Finally, the strip was treated with a chemiluminescent substrate (Thermo Scientific) for 5 min and developed by exposure to film for 5–20 s. The control MBP strip was probed with mouse monoclonal anti-MBP antibody (NEB).
Pip strip membranes
Manufactured by Thermo Fisher Scientific
Sourced in Germany
PIP strip membranes are a type of laboratory equipment used for protein detection and analysis. They provide a platform for the separation and identification of proteins based on their molecular weight or isoelectric point. The membranes are designed to be used in various protein analysis techniques, such as Western blotting and immunoblotting, to facilitate the visualization and quantification of specific proteins within a sample.
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2 protocols using pip strip membranes
1
Phosphoinositide Binding Protein Assay
2
PIP Strip Membrane-Based Peptide Binding Assay
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PIP strip membranes facilitate the analysis of phosphoinositide protein interactions by protein–lipid overlay assay. PIP strip membranes from Life Technologies (Darmstadt, Germany) were used (# P23750). GluA1 peptide was synthesized by Caslo (Lyngby, Denmark), and a fluorescein isothiocyanate (FITC) fluorochrome was conjugated to the N-terminus via an aminohexanoic acid (Ahx) linker. The GluA1 peptide sequence is (FITC)-Ahx-GALIEFCYKSRSESKRMKG. The first glycine was introduced for more flexibility of the peptide. The assay was performed as follows: the strip membrane was blocked with TBS-T + 3 % fatty-acid-free bovine serum albumin (BSA) (TBS-T = 10 mM Tris–HCl, pH 8.0; 150 mM NaCl; 0.1 % (v/v) Tween 20) and gently agitated for 1 h at room temperature. The peptide was solved in TBS-T + 3 % fatty-acid-free BSA and applied to the membrane. The membrane was incubated with 50 μg/ml peptide for 1 h at room temperature by gentle agitation. The washing of the membrane was performed three times with TBS-T + 3 % fatty-acid-free BSA for 10 min each time. For detection of fluorescence signal, the Molecular ImagerTM GelDoc from BioRad (Munich, Germany) was used. All incubation steps were performed in the dark.
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