For immunoprecipitation and PI3K lipid kinase assay, tissue lysates were prepared as described above. Immunoprecipitation was performed by mixing lysates (2 mg of protein) with 2 μg IRS-1 antibody (Millipore, cat. no. 05-1085). Following overnight incubation at 4 °C, immune complexes were captured by addition of protein-A-Sepharose beads (GE Healthcare) and incubation for an additional 1 h. Bead-associated immune complexes were subjected to lipid kinase assays. Lipid kinase assays were performed in a total volume of 50 μl in a buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 1 mM dithiothreitol, 5 mM MgCl2, 100 μM ATP (plus 0.1 μCi of [γ-32P]-ATP per assay) using 200 μg ml−1 phosphatidylinositol (Sigma) as a substrate. Reactions were incubated at 25 °C for 20 min and terminated by the addition of 100 μl of 0.1 M HCl and 200 μl of chloroform:methanol (1:1). The mixture was vortexed and the phases were separated by centrifugation at 10,000 × g for 2 min. The lower organic phase was spotted onto thin layer silica (TLC) gel-60 plates (Merck), which had been treated with 1% oxalic acid, 1 mM EDTA in water:methanol (6:4). TLC plates were developed in a solvent consisting of propanol-1:2 M acetic acid (13:7). Images of radiolabelled lipid products were captured using a Fuji FLA-2000 phosphorimager and analysed using ImageJ software.
Phosphatidylinositol
Manufactured by Merck Group
Sourced in United States, Germany
Phosphatidylinositol is a type of phospholipid found in the cell membrane of many organisms. It serves as a precursor for various signaling molecules involved in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatidylcholine, by Merck Group (6 mentions)
Phosphatidylserine, by Merck Group (4 mentions)
Phosphatidylethanolamine, by Merck Group (4 mentions)
Free glycerol detection reagent, by Merck Group (2 mentions)
Triolein, by Merck Group (2 mentions)
1 rac oleoylglycerol, by Merck Group (2 mentions)
Oleoyl coa, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Phosphatidic acid, by Merck Group (2 mentions)
Sphingomyelin, by Merck Group (2 mentions)
Lysophosphatidylcholine, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Methanol, by Merck Group (2 mentions)
Phosphatidylglycerol, by Merck Group (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Phosphatidylserine ps, by Merck Group (2 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Nefa kit, by Fujifilm (1 mentions)
Hi 76 0079, by Novo Nordisk (1 mentions)
1 oleoyl 2 hydroxy sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
14 protocols using phosphatidylinositol
1
Immunoprecipitation and PI3K Lipid Kinase Assay
2
Triolein Metabolism Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
[9,10-3H]Triolein was obtained from PerkinElmer Life Sciences. Triolein, phosphatidylcholine, phosphatidylinositol, 1(rac)-oleoylglycerol, oleoyl-CoA, and free glycerol detection reagents were purchased from Sigma. 1-Oleoyl-2-hydroxy-sn-glycero-3-phosphocholine was purchased from Avanti Polar Lipids Inc., Alabaster, AL, and the NEFA kit was from WAKO Diagnostics, Neuss, Germany. Hi76-0079 obtained from Novo Nordisk, Denmark, Atglistatin was a generous gift from R. Breinbauer (Graz University of Technology, Austria). The protein assay kit was obtained from Bio-Rad; Thermo Scientific, Rockford, IL was the source for the Pierce® BCA protein assay kit. The synthetic peptides were synthesized by Peptide Specialty Laboratories GmbH, Heidelberg, Germany.
3
Triolein Metabolism Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
If not stated otherwise, chemicals, antibiotics, and buffers were obtained from Sigma-Aldrich (St. Louis, MO) or Carl Roth GmbH (Karlsruhe, Germany); columns for protein purification were from GE Healthcare Life Sciences (Chicago, IL). The [9,10-3H] Triolein was obtained from PerkinElmer Life Sciences (Waltham, MA). Triolein, phosphatidylcholine, phosphatidylinositol, 1(rac)-oleoylglycerol, oleoyl-CoA, and free glycerol detection reagents were purchased from Sigma-Aldrich.
4
Optimized Microalgae Cultivation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lyophilized material of C. vulgaris (Midsona, Oslo, Norway) was purchased from a local health store. Lyophilized O. aurita was obtained from KissPlanet (Gembloux, Belgium). Kristalon Flower was purchased from Yara Norge as, Oslo, Norway. Sodium metasilicate pentahydrate was acquired from Skovly Engros as, Oslo, Norway. Kits for quantifying NO3, NO2, silicic acid, PO4 and NO4 were purchased from VWR, Radnor, Pennsylvania, USA. Dichloromethane (99.9%), methanol (99.8%), sulfuric acid (95–97%), hexane (99%), Sodium metasilicate pentahydrate (≥ 95%), sodium chloride, isopropanol and lipid standards of diacylglyceryl-trimethylhomoserine (1,2-dipalmitoyl-sn-glycero-3-O-4′-(N,N,N-trimethyl)-homoserine; DGTS), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) were purchased from Sigma Aldrich, St. Louis, Missouri, USA. Lipid standards of phosphatidylcholine (1,2-Dimyristoyl-sn-Glycero-3-Phosphatidylcholine; PC), phosphatidylglycerol (1,2-Dimyristoyl-sn-Glycero-3-phosphatidylglycerol Na Salt; PG), phosphatidylserine (1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine Na salt; PS), phosphatidylethanolamine (1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamine; PE), hydrogenated monogalactosyl diglyceride (MGDG), hydrogenated digalactosyl diglyceride (DGDG), ergosterol, triolein (TAG), diolein (DAG) and monolein (MAG) were purchased from Larodan AB, Solna, Sweden.
5
Tocopherol and Tocopheryl Phosphate Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RRR-α-tocopherol (αT), RRR-β-tocopherol (βT), RRR-γ-tocopherol (γT), RRR-δ-tocopherol (δT), and RRR-α-tocopheryl quinone (αTQ) (all from Cognis, Cincinnati, OH, USA) were dissolved in ethanol as 50 mM stock solutions and the concentrations confirmed spectrophotometrically. Stock solutions (50 mM) of α-tocopheryl phosphate (αTP), γ-tocopheryl phosphate (γTP) (all provided by Phosphagenics Ltd (Melbourne, Australia)) were prepared in ethanol or water [12] (link), [31] (link). Phosphatidylinositol was purchased from Sigma-Aldrich, Saint Louis, MO. Ritonavir (Moravek Biochemicals, CA, USA) was dissolved in ethanol as a 5 mg/mL stock.
6
Endogenous PIK3C3 Lipid Kinase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The endogenous PIK3C3 lipid kinase assay was performed according to a previous report.71 (link) Cells were lysed and immunoprecipitaed with anti-PIK3C3. Then, the immune complexes were incubated in a buffer (20 mM HEPES, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 mM ATP, 5 mM MnCl2, and 50 mM DTT, pH 7.4) containing 0.2 mg/ml phosphatidylinositol (Sigma, P5766) and 5 μCi 32P-ATP at 37 °C for 45 min. The kinase reactions were terminated by the addition of 20 μl of 8 M HCl and extracted with 160 μl chloroform:methanol (1:1). This extracted phospholipid products were separated on Silica Gel 60A (Merck, 115111). Plates were dried and followed by visualization with a Typhoon Imager (GE Healthcare Biosciences, Piscataway, NJ).
7
Cell Culture Reagents and Lipid Supplements
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, D-glucose, foetal bovine serum (FBS), L-glutamine, Trypsin, DNase I, corticosterone (CORT), Poly-L-Lysine, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), fibroblast growth factor (FGF), epidermal growth factor (EGF), and dimethyl sulfoxide were purchased from Sigma. Phosphatidylcholine (PC; from egg yolk), lysoPhosphatidylcholine (LPC; from egg yolk), phosphatidylserine (PS; from bovine brain), phosphatidylethanolamine (PE; from egg yolk), phosphatidylinositol (PI; from soy bean), phosphatidylglycerol (PG; from egg yolk), phosphatidic acid (PA; from egg yolk), sphingomyelin (SM; from egg yolk) and cardiolipin (CL; from bovine heart) were obtained in their respective salts from Sigma. B-27 supplement was obtained from Thermo Fisher Scientific.
8
Lipid Extraction and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dichloromethane, chloroform, hexane, methanol, isooctane, isopropanol, dimethylformamide, and acetonitrile, all of HPLC grade, were purchased from Labscan Ltd. (Dublin, Ireland) . Sodium sulfate anhydrous and sodium carbonate were obtained from Panreac Química SLU (Barcelona, Spain). Methyl tert-butyl ether was supplied by VWR International Eurolab SL (Barcelona, Spain). Sodium methoxide (95%), sodium citrate dehydrate, formic acid (98%), triethylamine (99.5%), the triacylglyceride (TAG) standards trinanoin and tritridecanoin, the free fatty acid standards, phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and sphingomyelin (SM) standards, as well as 5α-cholestane and cholesterol (Chol) were supplied by Sigma-Aldrich (St. Louis, MO). Reference samples with known composition, such as butter fat BCR-164 and BCR-519 (EU Commission; Brussels, Belgium) were from Fedelco SL (Madrid, Spain).
9
Lipid Standards for Biochemical Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Squalene, cholesterol, β-sitosterol, lanosterol, cycloartenol, campesterol, lathosterol, desmosterol, fucosterol, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, lysophosphatidylethanolamine, lysophosphatidylcholine and ceramide phosphorylethanolamine were purchased from Sigma Aldrich (St Louis, MO, USA). Zymosterol was purchased from Avanti Polar Lipids (Alabaster, AL, USA) and ergosterol was purchased from Supelco (Sigma-Aldrich Sweden AB, Stockholm, Sweden). All other growth media components were purchased from Sigma Aldrich (St Louis, MO, USA) and were of microbiological grade.
10
Transwell Permeability Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acetonitrile CHROMASOLV®, acycloguanosine, cholesterol (Chol), Fiske-SubbaRow reducer, phosphatidylinositol (PI), formic acid eluent additive for LC-MS, griseofulvin, glyceryl monooleate, indomethacin, maleic acid, methanol CHROMASOLV®, nadolol, potassium phosphate monobasic, sodium chloride, sodium dihydrogen phosphate, sodium hydroxide, sodium oleate, sodium taurocholate were obtained from Sigma-Aldrich, Steinheim, Germany. Ammonium molybdate, hydrogen peroxide, and disodiumhydrogenphosphat-dihydrat were purchased from Merck KGaA, Darmstadt, Germany.
Lipoid egg phospholipids (E-80), egg phophatidylcholine (PC), egg phophatidylethanolamine (PE), egg phophatidylserine (PS) were obtained from Lipoid, Ludwigshafen, Germany. Sulphuric acid was provided by May and Baker LTD Dagenham, England. All chemicals used in the experiments were of analytical grade.
Filter inserts (Transwell®, d = 6.5 mm) and plates were purchased from Corning Inc., Corning, New York, USA. The mixed cellulose ester filters (0.65 µm pore size) were purchased from Millipore, Billerica, Massachusetts, USA. Whatman® nucleopore track-etch membrane filters (0.4 µm, 0.8 µm and 1.2 µm pore size) were obtained from Whatman, part of GE Healthcare, Little Chalfont, UK.
Lipoid egg phospholipids (E-80), egg phophatidylcholine (PC), egg phophatidylethanolamine (PE), egg phophatidylserine (PS) were obtained from Lipoid, Ludwigshafen, Germany. Sulphuric acid was provided by May and Baker LTD Dagenham, England. All chemicals used in the experiments were of analytical grade.
Filter inserts (Transwell®, d = 6.5 mm) and plates were purchased from Corning Inc., Corning, New York, USA. The mixed cellulose ester filters (0.65 µm pore size) were purchased from Millipore, Billerica, Massachusetts, USA. Whatman® nucleopore track-etch membrane filters (0.4 µm, 0.8 µm and 1.2 µm pore size) were obtained from Whatman, part of GE Healthcare, Little Chalfont, UK.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!