NBD-Sphinganine and C18:0-NBD-ceramide were from Avanti Polar Lipids. Defatted-bovine serum albumin, FB1, a protease inhibitor cocktail, rabbit anti-CerS2, mouse anti-Flag, and mouse anti-GAPDH antibodies were from Sigma. Lipofermata was from Cayman Chemicals. A mouse anti-CerS2 antibody was from Santa Cruz and a rabbit anti-FATP2 antibody was from Proteintech. A rabbit anti-Calnexin antibody was purchased from Abcam. Horseradish peroxidase was from the Jackson Laboratory. An enhanced chemiluminescence detection system was from Thermo Scientific. 4x Laemmli sample buffer was from Bio-rad. Silica gel 60 TLC plates were from Merck. All the solvents were of analytical grade and were purchased from Bio-Lab. Anti-DYKDDDDK G1 Affinity Resin, pcDNA 3.1(+) vectors containing FATP1-6, and FATP2b with a C-terminus Flag-tag were from GenScript.
Nbd sphinganine
Manufactured by Avanti Polar Lipids
NBD-Sphinganine is a fluorescent lipid analog that can be used as a probe for the study of sphingolipid metabolism and trafficking. It consists of a fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group attached to the C4 position of sphinganine.
Automatically generated - may contain errors
Lab products found in correlation
Defatted bovine serum albumin, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (3 mentions)
Silica gel 60 thin layer chromatography plate, by Merck Group (2 mentions)
Fatty acyl coa, by Avanti Polar Lipids (2 mentions)
Imagequant tl, by GE Healthcare (2 mentions)
Mouse anti gapdh, by Merck Group (1 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Silica gel 60 tlc plate, by Merck Group (1 mentions)
Mouse anti flag, by Merck Group (1 mentions)
Lipofermata, by Cayman Chemical (1 mentions)
Rabbit anti fatp2 antibody, by Proteintech (1 mentions)
Rabbit anti calnexin antibody, by Abcam (1 mentions)
Anti tubulin antibody, by Merck Group (1 mentions)
Anti ha, by Merck Group (1 mentions)
Anti cers6 antibodies, by Santa Cruz Biotechnology (1 mentions)
Protein a agarose beads, by Santa Cruz Biotechnology (1 mentions)
Anti cers2, by Merck Group (1 mentions)
Ecl detection system, by Cyanagen (1 mentions)
5 protocols using nbd sphinganine
1
Lipid Analysis in Cell Signaling
2
Lipid Biosynthesis Pathway Probing
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NBD-Sphinganine and fatty acyl CoAs were from Avanti Polar Lipids. Defatted-bovine serum albumin, a protease inhibitor cocktail, anti-HA, anti-CerS2, and anti-tubulin antibodies were from Sigma-Aldrich. Protein A agarose beads and anti-CerS6 antibodies were from Santa Cruz. Horseradish peroxidase was from the Jackson Laboratory. An ECL detection system was from Cyanagen. Silica gel 60 thin layer chromatography plates were from Merck. All solvents were of analytical grade and purchased from Bio-Lab.
3
In vitro assay for ceramide synthase
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NBD-sphinganine and palmitoyl-CoA (C16-CoA) were from Avanti Polar Lipids (Alabaster, AL). Defatted-bovine serum albumin, a protease inhibitor cocktail and an anti-CerS6 antibody were from Sigma-Aldrich (St. Louis, MO). Horseradish peroxidase was from the Jackson Laboratory (Bar Harbor, ME). An enhanced chemiluminescence (ECL) detection system was from Cyanagen (Bologna, Italy). An anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody and silica gel 60 thin layer chromatography plates were from Merck (Billerica, MA). All solvents were of analytical grade and purchased from Bio-Labs (Jerusalem, Israel).
4
CerS2 Activity Assay in Cells and Tissues
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CerS2 activity was assayed in homogenates of HEK293 cells, mouse liver, and kidney as previously described.54 (link) Briefly, cell and tissue homogenates (40 μg of total protein) were incubated at 37°C for 25 min with 15 μM NBD sphinganine, 50 μM 24:1-CoA (Avanti Polar Lipids), and 20 μM defatted bovine serum albumin (Sigma-Aldrich). Reactions were terminated by addition of chloroform/methanol (1:2, v/v), and lipids were extracted using the Bligh-Dyer procedure. Lipid extracts were dried under N2, dissolved in chloroform/methanol (9:1, v/v), and separated by thin-layer chromatography using chloroform/methanol/2 M NH4OH (40:10:1, v/v/v) as the developing solvent. NBD-labeled lipids were visualized using an Amersham Typhoon 5 biomolecular imager (GE Healthcare) and quantified by ImageQuant TL (GE Healthcare).
5
Sphinganine Acylation Activity Assay
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Cell homogenates were prepared in 20 mM HEPES-KOH, pH 7.2, 25 mM KCl, 250 mM sucrose, and 2 mM MgCl 2 containing a protease inhibitor mixture. Protein was determined using the BCA reagent (Thermo Fisher Scientific). Samples were incubated with 15 µM NBD-sphinganine (Avanti Polar Lipids), 20 µM defatted BSA (Sigma-Aldrich), and 50 µM fatty acyl-CoA (Avanti Polar Lipids) in a 20 µl reaction volume. CerS (40 µg protein, 25 min reaction time) was assayed using C24.1-CoA and Cer5/6 (5 µg protein, 5 min reaction time) assayed using C16-CoA. Reactions were terminated by chloroform/methanol (1:2, v/v) and lipids extracted. Lipids were dried under N 2 , resuspended in chloroform/methanol (9:1, v/v), and separated by TLC (Merck) using chloroform/methanol, 2M NH 4 OH (40:10:1, v/v/v) as the developing solvent. NBD-labeled lipids were visualized using an Amersham Typhoon5 imager and quantified by ImageQuantTL (GE Healthcare, Chalfont St Giles, UK). All solvents were of analytical grade and were purchased from Bio-Lab (Jerusalem, Israel).
Cell homogenates were prepared in 20 mM HEPES-KOH, pH 7.2, 25 mM KCl, 250 mM sucrose, and 2 mM MgCl 2 containing a protease inhibitor mixture. Protein was determined using the BCA reagent (Thermo Fisher Scientific). Samples were incubated with 15 µM NBD-sphinganine (Avanti Polar Lipids), 20 µM defatted BSA (Sigma-Aldrich), and 50 µM fatty acyl-CoA (Avanti Polar Lipids) in a 20 µl reaction volume. CerS (40 µg protein, 25 min reaction time) was assayed using C24.1-CoA and Cer5/6 (5 µg protein, 5 min reaction time) assayed using C16-CoA. Reactions were terminated by chloroform/methanol (1:2, v/v) and lipids extracted. Lipids were dried under N 2 , resuspended in chloroform/methanol (9:1, v/v), and separated by TLC (Merck) using chloroform/methanol, 2M NH 4 OH (40:10:1, v/v/v) as the developing solvent. NBD-labeled lipids were visualized using an Amersham Typhoon5 imager and quantified by ImageQuantTL (GE Healthcare, Chalfont St Giles, UK). All solvents were of analytical grade and were purchased from Bio-Lab (Jerusalem, Israel).
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