Cholesterol

Manufactured by Steraloids

Cholesterol is a laboratory product that serves as a reference standard and analytical reagent. It is a naturally occurring steroid compound found in the cell membranes of various organisms. Cholesterol is commonly used for the development and calibration of analytical procedures, as well as for quality control purposes in laboratories.

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Lab products found in correlation

Propofol, by LGC (2 mentions) Propofol, by Steraloids (2 mentions) Phospholipid, by Avanti Polar Lipids (2 mentions) L glutamate, by Merck Group (2 mentions) Alexa fluor 594 and alexa fluor 488 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions) Vimentin, by Abcam (1 mentions) 25 hydroxycholesterol, by Steraloids (1 mentions) 1 2 3h cholesterol, by PerkinElmer (1 mentions)

6 protocols using cholesterol

Recombinant CYP27A1 Ligand Binding

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Human recombinant CYP27A1 was expressed and purified as described by Mast et al. (2006) (22 ). Propofol (Toronto Research Chemicals Inc.) and cholesterol (Steraloids Inc.) binding to CYP27A1 was analysed in vitro according to the method described by Lam et al. (2018) (19 (link)), using Propofol concentrations of 20, 40 and 100 μM and cholesterol concentrations up to 3.0 μM.

Claesen J.L., Koomen E., Schene I.F., Jans J.J., Mast N., Pikuleva I.A., van der Ham M., de Sain‐van der Velden M.G, & Fuchs S.A. (2020). Misdiagnosis of CTX due to propofol: The interference of total intravenous propofol anaesthesia with bile acid profiling. Journal of Inherited Metabolic Disease, 43(4), 843-851.

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Characterizing Propofol and Cholesterol Binding to CYP27A1

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Human recombinant CYP27A1 was expressed and purified as described by Mast et al.22 Propofol (Toronto Research Chemicals, Inc) and cholesterol (Steraloids, Inc) binding to CYP27A1 was analysed in vitro according to the method described by Lam et al,19 using Propofol concentrations of 20, 40, and 100 μM and cholesterol concentrations up to 3.0 μM.

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Cholesterol and Lipid Radiolabeling Protocols

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Cholesterol and 25-hydroxyCholesterol were purchased from Steraloids (Newport, RI). [1,2-³H]Cholesterol, 25-[26,27-³H]hydroxyCholesterol and [³²P]PO₄ were purchased from PerkinElmer Life Sciences (Boston, MA). [¹⁴C]-Dipalmitoyl-phosphatidylcholine was purchased from American Radiolabeled Chemicals (St. Louis, MO). Phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). Antibodies against giantin and polypeptide N-acetylgalactosaminyltransferase (GALNT) (BioLegend, San Diego CA), vimentin (Abcam, Cambridge UK) and V5 (BioRad, Raleigh NC) were used for immunofluorescence microscopy and immunoblotting. Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Molecular Probes, Eugene OR) and IRDye 680LT- and IRDye 800CW-conjugated secondary antibodies (LI-COR Biosciences, Lincoln NE) were used for immunofluorescence and immunoblotting, respectively.

Pietrangelo A, & Ridgway N.D. (2019). Phosphorylation of a serine/proline-rich motif in oxysterol binding protein-related protein 4L (ORP4L) regulates cholesterol and vimentin binding. PLoS ONE, 14(3), e0214768.

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Cholesterol Metabolism Enzyme Assay

Cited 1 time

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(S)-EFV ((S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-1,4-dihydro-2H-benzo[d][1,3]oxazin-2-one) and all EFV metabolites, as well as analogs, were purchased from Toronto Research Chemicals (Toronto, ON, Canada) and were >95% pure according to the manufacturer’s certificates of analysis (Figures S2–S11). ₁H NMR and ₁₉F NMR spectra of these compounds conformed to their structures. L-Glutamate was from MilliporeSigma (St. Louis, MO) and a 2 mM stock solution was prepared in water. Cholesterol was obtained from Steraloids (Newport, RI), and 24-hydroxy-[25,26,26,26,27,27,27-₂H₇]-Cholesterol was from Medical Isotopes (Pelham, NH). Cholesterol was added from a 1 mM stock in 4.5%, w/v, aqueous 2-hydroxypropyl-β-cyclodextrin (HPCD) and deuterated 24-hydroxyCholesterol was added from a 0.1 mM stock in methanol. Human truncated Δ(2–50)CYP46A1 with a four-histidine tag on the C terminus and rat cytochrome P450 oxidoreductase were expressed in Escherichia coli and purified as described.60 (link),61 (link) The F405A Δ(2–50)CYP46A1 mutant was generated by using an in vitro QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA) according to the instructions. The correct generation of the desired mutation and absence of undesired mutations were confirmed by nucleotide sequencing of the entire CYP46A1 coding region as well as by the restriction analysis.

Mast N., Verwilst P., Wilkey C.J., Guengerich F.P, & Pikuleva I.A. (2019). In Vitro Activation of Cytochrome P450 46A1 (CYP46A1) by Efavirenz-Related Compounds. Journal of Medicinal Chemistry, 63(12), 6477-6488.

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Cholesterol Oxidation Assay Protocol

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Cholesterol was obtained from Steraloids and phospholipids from Avanti Polar Lipids. HPCD (ave. MW = 1396) and methyl-β-cyclodextrin (ave. MW = 1310) were purchased from Sigma-Aldrich. Cholesterol oxidase (EC 1.1.3.6; Streptomyces sp; reported activity of 43.9 U/mg protein) was obtained from EMD. The BCA protein assay kit was from Pierce.

Lange Y., Ye J, & Steck T.L. (2014). Essentially All Excess Fibroblast Cholesterol Moves from Plasma Membranes to Intracellular Compartments. PLoS ONE, 9(7), e98482.

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Enzymatic Synthesis of 24-Hydroxycholesterol

Cited 1 time

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(S)-EFV ((S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-1,4-dihydro-2H-benzo[d][1,3]oxazin-2-one) and
all EFV metabolites, as well as analogs, were purchased from Toronto
Research Chemicals (Toronto, ON, Canada) and were >95% pure according
to the manufacturer’s certificates of analysis (Figures S2–S11). ¹H NMR and ¹⁹F NMR spectra of these compounds conformed to their structures. l-Glutamate was from MilliporeSigma (St. Louis, MO), and a 2
mM stock solution was prepared in water. Cholesterol was obtained
from Steraloids (Newport, RI), and 24-hydroxy-[25,26,26,26,27,27,27-²H₇]-Cholesterol was from Medical Isotopes (Pelham,
NH). Cholesterol was added from a 1 mM stock in 4.5%, w/v, aqueous
2-hydroxypropyl-β-cyclodextrin (HPCD), and deuterated 24-hydroxyCholesterol
was added from a 0.1 mM stock in methanol. Human truncated Δ(2–50)CYP46A1
with a four-histidine tag on the C terminus and rat cytochrome P450
oxidoreductase were expressed in Escherichia coli and purified as described.^{60 (link),61 (link)} The F405A Δ(2–50)CYP46A1
mutant was generated by using an in vitro QuikChange site-directed
mutagenesis kit (Stratagene, San Diego, CA) according to the instructions.
The correct generation of the desired mutation and absence of undesired
mutations were confirmed by nucleotide sequencing of the entire CYP46A1
coding region as well as by the restriction analysis.

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