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.
Fatty acyl coa
Manufactured by Avanti Polar Lipids
Fatty acyl CoAs are key intermediates in lipid metabolism. They are the activated forms of fatty acids, which are essential for various cellular processes such as energy production, membrane synthesis, and signal transduction. Fatty acyl CoAs serve as substrates for a wide range of enzymatic reactions, playing a crucial role in the regulation and maintenance of cellular lipid homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Ecl detection system, by Cyanagen (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Nbd sphinganine, by Avanti Polar Lipids (2 mentions)
Defatted bsa, by Merck Group (2 mentions)
Defatted bovine serum albumin, by Merck Group (1 mentions)
Silica gel 60 thin layer chromatography plate, by Merck Group (1 mentions)
Anti tubulin antibody, by Merck Group (1 mentions)
Anti cers2, by Merck Group (1 mentions)
Protein a agarose beads, by Santa Cruz Biotechnology (1 mentions)
Anti cers6 antibodies, by Santa Cruz Biotechnology (1 mentions)
Anti ha, by Merck Group (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Silica gel 60 tlc plate, by Merck Group (1 mentions)
Polyethyleneimine, by Merck Group (1 mentions)
Nadph, by Merck Group (1 mentions)
Db 5 column, by Agilent Technologies (1 mentions)
Amersham typhoon5 imager, by GE Healthcare (1 mentions)
Bca reagent, by Thermo Fisher Scientific (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
4 protocols using fatty acyl coa
1
Lipid Biosynthesis Pathway Probing
2
Lipid Extraction and Analysis Protocol
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NBD-Sphinganine (NBD-Sph) and fatty acyl-CoAs were from Avanti Polar Lipids (Alabaster, AL). Defatted BSA, a protease inhibitor mixture, and polyethyleneimine were from Sigma. An ECL detection system and a BCA reagent kit were from Cyanagen (Bologna, Italy). Silica gel 60 TLC plates were from Merck (Billerica, MA). All solvents were of analytical grade and were purchased from Bio-Lab (Jerusalem, Israel).
3
Fatty Acid Reductase Activity Assay
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Recombinant FarO and CPR were assayed for reductase activity by incubating infected Sf9 cell lysate preparations with NAD(P)H and fatty acid (Sigma-Aldrich) or fatty acyl-CoA (Avanti Polar Lipids) substrates. Briefly, cell lysate supernatants in CLB were incubated in 100mM Tris HCl pH 7.0, 100 μM DTT, 0.5 mM PMSF and 10 μl of protease inhibitor cocktail (Sigma) supplemented with 120–150 μM 26:0–CoA or 24:0–CoA and 2.3 mM NADH (Fisher Scientific) or NADPH (Sigma Aldrich). All listed concentrations are final in 600–1000 μl reaction volumes. Samples were incubated at 30°C for 2 h and then extracted twice with hexane:ether (50/50, v/v) into glass vials. Samples were dried down to completion under N2 gas, resuspended in pure hexane and analyzed by gas chromatography using either a DB-5 column (Agilent) or a Shimadzu non-polar polysiloxane column, (catalog number: 220-94536-01, phase: SHR5XLB) with the following profile: injector temperature 150°C, FID temperature 300°C; program:160°C for 0.2 min, ramp to 265°C at 15°C/min, ramp to 295°C at 5°C/min, and hold for 5 min at 295°C.
4
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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