For this protein–lipid overlay assay, PIP Membrane Strips (Invitrogen™ P23751) were used to determine the GST-LdSNXi full-length lipid specificity. Each strip, after an initial blocking step (1 h, RT) with 5% (w/v) bovine serum albumin (BSA) in TBST [Tris-buffered saline (TBS; 50 mM Tris-Cl, pH 7.5, 150 mM NaCl) with 0.05% (v/v) Tween-20], was overlaid with equimolar solutions of either purified recombinant GST-LdSNXi [50 ng/mL in 5% (w/v) BSA, TBST] or 17 ng/mL of purified GST (negative control) followed by a 3 h incubation at RT. Subsequently, the strips were washed three times (RT, 10 min) with TBST followed by a second protein-binding step (4 °C, overnight) with GST-LdSNXi [50 ng/mL, in 5% (w/v) BSA, TBST] or GST (17 ng/mL) and 3 washings with TBST. The bound protein was further detected by the a-GST (0.5 μg/mL) rabbit pAb (A5800; Invitrogen, Waltham, MA, USA) following the Western blot and development by ECL protocols described below.
Pip membrane strips
Manufactured by Thermo Fisher Scientific
The PIP Membrane Strips are a laboratory equipment product designed for protein-protein interaction (PPI) studies. The strips provide a simple and efficient method for detecting and analyzing protein-protein interactions in a high-throughput manner. The core function of the PIP Membrane Strips is to facilitate the identification and characterization of protein-protein interactions, enabling researchers to better understand complex biological processes.
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2 protocols using pip membrane strips
1
Lipid Specificity Determination via GST-LdSNXi Overlay
2
Protein-Lipid Binding Assay Protocol
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Protein–lipid overlay assays were performed as described previously (Lee and Wu, 2012 (link)). Briefly, the PIP membrane strips (Invitrogen, Cat. no. P23751) were first blocked using TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) with 3% fatty-acid-free BSA (Sigma, cat. no. A7030) at 23°C for 1 h with shaking. Purified proteins were incubated with the membrane at a final concentration of 50 nM for 1 h at 23°C or overnight at 4°C. The membrane was then washed with TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) to remove the unbound protein. Lipid bindings were examined by immunoblot with anti-His antibody (Clontech, cat. no. 631212; 1:20,000 dilution) as primary antibody and anti-mouse IgG as secondary antibody (1:5000 dilution) in TBS + 3% fatty-acid-free BSA.
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