Sphingo strips (Echelon Biosciences Inc., Salt Lake City, UT) were probed with various proteins according to the manufacturer’s instructions. Briefly, the Sphingo strips were blocked at RT for 2 h in blocking buffer (10 mM Tris, 150 mM NaCl, 0.1% Tween-20 and 3% BSA, pH 8.0) and washed three times with wash buffer (10 mM Tris, 150 mM NaCl and 0.1% Tween-20, pH 8.0). Purified BmTFF3 was incubated with the Sphingo strips at RT for 1 h in blocking buffer at a concentration of 2 μg ml−1. Then, the protein solution was removed and washed three times with wash buffer. A rabbit anti-BmTFF3 polyclonal antibody (1:1000 dilution) was used as the primary antibody, and HRP-conjugated goat anti rabbit secondary antibodies (1:5000 dilution) were used as the secondary antibody. Binding was detected with the SuperSignal WestPico chemiluminescence substrate (Invitrogen).
Sphingo strips
Manufactured by Echelon Biosciences
Sourced in United States
Sphingo Strips are a lab equipment product from Echelon Biosciences. The Sphingo Strips are used for the separation and detection of sphingolipids. They provide a simple and efficient method for the analysis of sphingolipid species in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Membrane lipid strip, by Echelon Biosciences (1 mentions)
Las 3000 detection system, by Fujifilm (1 mentions)
Anti gst antibody, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico, by Fujifilm (1 mentions)
Hrp conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Pip strip, by Echelon Biosciences (1 mentions)
4 protocols using sphingo strips
1
Sphingo Strip Protein Binding Assay
2
Protein-Lipid Interaction Assay
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Protein-lipid overlay assay was performed for membrane lipids and sphingolipids with Membrane Lipid Strips (P-6002; Echelon Biosciences, Salt Lake City, USA) and Sphingo Strips (S-6000; Echelon Biosciences), respectively according to the manufacturer’s instructions. In brief, the strip was incubated with the blocking buffer (PBS with 0.1% Tween-20 and 3% fatty acid-free BSA) for 1 hr at room temperature, and 1 μg/ml of recombinant murine AM protein (MBS2012437; MyBioSource) was added to the strip for 1 hr. Primary anti-mouse AM antibody (sc-366637; Santa Cruz Biotechnology), secondary HRP-conjugated anti-rabbit antibody, and Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Billerica, USA) were used to detect positive spots. Signals were detected with ImageQuant LAS4000 Mini (GE Healthcare Life Sciences).
3
Lipid Binding Assay for Ly49Q Protein
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The lipid binding ability of Ly49Q was examined using Sphingo Strips (Echelon Biosciences Inc.) according to the manufacturer’s instructions. In brief, Shingo Strip membranes were incubated with GST-tagged Ly49Q proteins or control GST [5 µg/ml in tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA)] at 4°C overnight. After washing with TBS-T to remove unbound GST-tagged proteins, Shingo Strip membranes were further incubated with anti-GST antibody (Santa Cruz Biotechnology, Inc.) and subsequently HRP-conjugated anti-mouse IgG (Jackson Laboratory). HRP was detected with Supersignal West Femto or Supersignal West Pico chemiluminescent substrates with the LAS-3000 detection system (FUJIFILM Co., Tokyo, Japan).
4
Lipid Binding Assay of GST and GST-MAM
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PIP-Strips and Sphingo-Strips (Echelon Biosciences) were used to test the lipid-binding properties of GST and GST-MAMHS. Each membrane was spotted with 100 pmol of each lipid species. Membranes were blocked with blocking buffer (0.1% v/v Tween 20, 5% skim milk in PBS) for 1 h at room temperature with gentle shaking. 10 μm protein was added and incubated for 1 h with shaking. Strips were washed with washing buffer (0.1% v/v Tween 20 in PBS) three times for 10 min each. GST antibody (1:1000 in blocking buffer) was applied and incubated for 1 h at room temperature with shaking. Membranes were washed with washing buffer for 10 min; this step was repeated three times. The secondary-anti mouse IgG-HRP antibody (1:5000 in blocking buffer) was added and incubated for 1 h at room temperature with shaking. Strips were then washed with washing buffer three times for 10 min each and developed using Clarity ECLTM Western substrate.
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