The assay was according to the lipid-binding analysis method by Munnik and Wierzchowiecka (2013) (link). Ceremides, ganglioside GM3, galactocerebrosides, sulfatide, D-sphingosine, L-α-phosphatidylinositol and L-phosphatidylinositol (Sigma-Aldrich) were suspended in resuspension buffer (CHCl3/MeOH/H2O=20:9:1, v/v), and diluted to gradient concentration by spotting buffer (CHCl3/MeOH/50 mM HCl/Ponceau S=250:500:200:2, v/v). The lipids were spotted on PVDF membrane strips and dried at room temperature in the dark. The membranes were blocked in fat blot buffer (50 mM Tris–HCl, 150 mM NaCl, pH 7.5) containing 3% fatty acid-free BSA for 1 h at room temperature, and incubated with SMMC-7721 cell lysates (protein 5 μg/ml) in blocking buffer-0.1% Tween overnight at 4°C. After three washes with fat blot buffer-0.1% Tween, the membranes were incubated with anti-SIN3B antibody and goat anti-rabbit IgG HRP secondary antibody (Jackson ImmunoReseach Lab, Inc.). The blots were visualized using Immunobilon Western (Millipore).
L phosphatidylinositol
Manufactured by Merck Group
L-phosphatidylinositol is a laboratory product used for research purposes. It is a phospholipid compound that is a component of cellular membranes. This product can be used in various research applications involving the study of cellular processes and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
D sphingosine, by Merck Group (1 mentions)
Immunobilon western, by Merck Group (1 mentions)
L α phosphatidylinositol, by Merck Group (1 mentions)
Cholesterol, by Merck Group (1 mentions)
1 2 dioleoyl sn glycero 3 phosphoethanolamine n lissamine rhodamine b sulfonyl ammonium salt rhodamine pe, by Avanti Polar Lipids (1 mentions)
L phosphatidylethanolamine, by Merck Group (1 mentions)
L phosphatidyl l serine, by Merck Group (1 mentions)
Sphingomyelin, by Merck Group (1 mentions)
2 protocols using l phosphatidylinositol
1
Lipid-binding Analysis in SMMC-7721 Cells
2
Preparation and Characterization of Lipid Vesicles
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The lipids L-␣-phosphatidylcholine (PC, >98%), cholesterol (CHOL, >99%), L-␣-phosphatidylethanolamine, (PE, >99%), sphingomyelin (SM, >97%), L-␣-phosphatidyl-l-serine (PS, >97%), and L-␣-phosphatidylinositol (PI, >50%) were purchased as a powder from Sigma-Aldrich, and dissolved in chloroform. Urease type III from C. ensiformis (Jack bean) was obtained from Sigma-Aldrich.
The 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(Lissamine rhodamine B sulfonyl) (ammonium salt) -(Rhodamine-PE, >99%) was purchased from Avanti Polar Lipids. Triton X-100 was purchased from the InLab Company.
The 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(Lissamine rhodamine B sulfonyl) (ammonium salt) -(Rhodamine-PE, >99%) was purchased from Avanti Polar Lipids. Triton X-100 was purchased from the InLab Company.
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