Extracellular buffer with glucose (ECB-glucose) was composed of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2 and 10 mM glucose (pH 7.4). Tris buffer for separations was 25 mM tris(hydroxymethyl)aminomethane (Tris) (pH 8.4). Small unilamellar vesicles were prepared by rehydrating a film of 2 mg egg phosphatidylcholine (Avanti Polar Lipids) in 2 mL of 10 mM Tris and 150 mM NaCl (pH 7.4) with sonification (S-250A, Branson) at a 20% duty cycle for 30 min. The resulting vesicles were stored at 4 °C for at least 2 h and up to 2 weeks prior to use. The reporter peptide, YSYQMALTPVV(K-FAM)TL, was synthesized by Anaspec and stored as a 2 mM stock solution in DMSO at −20 °C under desiccation. Tosedostat (CHR-2797) was obtained from SelleckChem and stored as a 10 mM or 80 mM stock solution in DMSO at −20 °C under desiccation.
Egg phosphatidylcholine
Manufactured by Avanti Polar Lipids
Sourced in United States
Egg phosphatidylcholine is a natural lipid compound derived from egg yolks. It is a major component of cell membranes and plays a crucial role in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Roche (3 mentions)
Hepes, by Merck Group (3 mentions)
Dspe peg2000, by Avanti Polar Lipids (3 mentions)
Igg agarose resin, by GE Healthcare (2 mentions)
Anti dpm1 antibody, by Abcam (2 mentions)
Sm 2 biobeads and p6 biogel resin, by Bio-Rad (2 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Calcein, by Merck Group (2 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (2 mentions)
Cholic acid, by Merck Group (2 mentions)
Bodipy cholesterol, by Avanti Polar Lipids (2 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (2 mentions)
Egg phosphatidylethanolamine, by Avanti Polar Lipids (2 mentions)
Dogs nta ni, by Avanti Polar Lipids (2 mentions)
Bovine heart cardiolipin, by Avanti Polar Lipids (2 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (2 mentions)
Centrifugal concentrator, by Merck Group (1 mentions)
Valinomycin, by Merck Group (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Lds loading buffer, by Thermo Fisher Scientific (1 mentions)
36 protocols using egg phosphatidylcholine
1
Preparation of Phospholipid Vesicles and Peptide Reagents
2
Liposome Preparation with Fluorescent Probe
3
Purification and Characterization of Membrane Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isopropyl-β-d -thiogalactopyranoside (IPTG), Tris(2-carboxyethyl)phosphine (TCEP), n-dodecyl-β-d -maltopyranoside (DDM) and precast SDS–PAGE gels were from Generon. All aromatic acids, l -arabinose, cholesteryl hemisuccinate Tris salt (CHS), 0.4–0.6 mm acid-washed glass beads, valinomycin and Proteinase K-agarose were from Sigma–Aldrich. HisTrap columns, PD-10 and PD SpinTrap G-25 gel filtration columns, Superdex 200 10/300 GL column, size exclusion column standards, and nitrocellulose membrane were from GE Healthcare. Chemiluminescence reagents (LumiGLO) were from Cell Signaling Technologies. E. coli strain BL21-AI, LDS loading buffer, V5-HRP, pyranine and gels and reagents for blue native PAGE were from Life Technologies. Centrifugal concentrators were from Millipore. The detergent compatible Lowry assay was purchased in kit format from ThermoFisher. E. coli polar lipid extract and egg phosphatidylcholine were from Avanti Polar Lipids.
4
Yeast Protein Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analytical-grade reagents were purchased from Sigma-Aldrich unless stated otherwise.
Antibiotics were from Sigma-Aldrich, Invivogen or Invitrogen. Other reagents/materials were obtained as follows: D-2-[ 3 H]-mannose (20-30 Ci/mmol) (American Radiolabeled Chemicals Inc.), Micro BCA protein assay kit (Thermo Fisher Scientific), Triton X-100 (Roche), IgG-Agarose resin (GE Healthcare), anti-TAP antibody (Genescript), anti-Dpm1 antibody (Abcam), SM-2 Biobeads and P6-Biogel resin (Biorad) and egg phosphatidylcholine (Avanti Polar Lipids). Yeast strains used in this study are listed in Table S5.
Antibiotics were from Sigma-Aldrich, Invivogen or Invitrogen. Other reagents/materials were obtained as follows: D-2-[ 3 H]-mannose (20-30 Ci/mmol) (American Radiolabeled Chemicals Inc.), Micro BCA protein assay kit (Thermo Fisher Scientific), Triton X-100 (Roche), IgG-Agarose resin (GE Healthcare), anti-TAP antibody (Genescript), anti-Dpm1 antibody (Abcam), SM-2 Biobeads and P6-Biogel resin (Biorad) and egg phosphatidylcholine (Avanti Polar Lipids). Yeast strains used in this study are listed in Table S5.
5
Liposomal Delivery of Luciferin for Inflammation Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 9
Aliquots of lipids (2.6 mM of egg phosphatidylcholine (Avanti Polar Lipids, Inc.) and 0.1 mM of lipidated inhibitor) supplied as chloroform solutions are placed into vials to form thin films by removing chloroform by evaporation under vacuum. Dry films are then hydrated by adding of D-luciferine in PBS (15 mg/ml). Dispersions are homogenized with vortex mixing and extruded under pressure through polycarbonate filters of decreasing pore diameter 0.1 μm using extruder. Targeted delivery of liposomes labeled by lipidated inhibitor carrying D-luciferin into transgenic mouse expressing luciferase (FVB.luctg/+) is shown in
6
Preparation of Lipid-Inhibitor Dispersions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 4
Aliquots of lipids (2.6 mM of egg phosphatidylcholine (Avanti Polar Lipids, Inc.) and 0.1 mM of lipidated inhibitor) supplied as chloroform solutions are placed into vials to form thin films by removing chloroform by evaporation under vacuum. Dry films are then hydrated by adding of the 0.1 mM phosphate buffer pH 6.0. Dispersions are homogenized with vortex mixing and then emulsified by sonication in bath sonicator during 20 min.
7
Brominated Lipids and Detergents Effects on SERCA1a
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brominated phosphatidylcholine (BrPC, 1,2-di-(9,10-dibromo)stearoyl-sn-glycero-3-phosphocholine, #850366) and egg phosphatidylcholine (#840051) were from Avanti Polar Lipids, DDM (n-dodecyl-β-D-maltoside) and C12E8 (octaethylene glycol monododecyl ether) were from Anatrace, and other chemicals were of standard grade. SERCA1a-containing sarcoplasmic reticulum (SR) membranes (with lipid and protein contents of about 0.5 g lipid per g protein [15 (link)]) were prepared from rabbit skeletal muscle as described [16 (link)]. Streptavidin-purified yeast Drs2p/Cdc50p complex was also prepared as described [17 (link)]. Brominated DDM (BrDDM, 5,6-dibromo-dodecyl-β-D-maltoside) was synthesized as described [18 ]; its critical micelle concentration (cmc), measured according to the methyl orange method [19 (link)], was found only 20% higher than that for DDM (see similar result with 7–8 BrDDM in [14 ]), and BrDDM affected SERCA1a ATPase activity in a manner very similar to DDM (S1 Fig ). Two different buffers were used, either buffer A (100 mM KCl, 1 mM MgCl2, 50 mM Tes-Tris at pH 7.5 and 0.1 mM CaCl2), or buffer B (100 mM KCl, 5 mM MgCl2, 50 mM Mops-Tris at pH 7 and contaminating Ca2+ from demineralized water, a few micromolar).
8
Cell Surface Glycan Labeling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analytical-grade reagents were purchased from Sigma-Aldrich unless stated otherwise. Antibiotics were from Sigma-Aldrich, Invivogen or Invitrogen. Other reagents/materials were obtained as follows: D-2-[3H]-mannose (20–30 Ci/mmol) (American Radiolabeled Chemicals Inc.), Micro BCA protein assay kit (Thermo Fisher Scientific), Triton X-100 (Roche), IgG-Agarose resin (GE Healthcare), anti-TAP antibody (Genescript), anti-Dpm1 antibody (Abcam), SM-2 Biobeads and P6-Biogel resin (Biorad) and egg phosphatidylcholine (Avanti Polar Lipids). Yeast strains used in this study are listed in Table S5 .
9
Recombinant Sticholysin I and Hemolysins Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant wild-type Stichodactyla helianthus sticholysin I (StIr) and its mutant StI W111C were isolated as described [21] (link), [31] (link). S. aureus alpha-hemolysin (αHL) was from Sigma, and gamma-hemolysin (HlgA/HlgB) was a kind gift of G. Prévost.
Calcein was obtained through Sigma Chemical Co. and Triton X-100 from Merck. Lipids Egg phosphatidylcholine (PC), 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), 1,2 diphytanoyl-sn-glycerophosphocholine (DPhPC), porcine brain sphingomyelin (SM) and egg SM were from Avanti Polar Lipids (Alabaster). The 1-lauroyl-2-(1′pyrenebutyroyl)-sn-glycero-3-phosphocholine (pyPC) was kindly donated by Dr. P. Muller. Ethylene glycol (EG) was from Sigma. Polyethylene glycol 400 (PEG 400) was from Jansen and PEG200, PEG600 and PEG 1000 were from Fluka.
Calcein was obtained through Sigma Chemical Co. and Triton X-100 from Merck. Lipids Egg phosphatidylcholine (PC), 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), 1,2 diphytanoyl-sn-glycerophosphocholine (DPhPC), porcine brain sphingomyelin (SM) and egg SM were from Avanti Polar Lipids (Alabaster). The 1-lauroyl-2-(1′pyrenebutyroyl)-sn-glycero-3-phosphocholine (pyPC) was kindly donated by Dr. P. Muller. Ethylene glycol (EG) was from Sigma. Polyethylene glycol 400 (PEG 400) was from Jansen and PEG200, PEG600 and PEG 1000 were from Fluka.
10
Cholesterol Efflux Assay Using Fluorescent Probes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at 2×104 cells per well in black 96-well plates and incubated for 24 h. Cells were treated with or without cholesterol for 1 h and replaced with serum-free medium for 24 h before being treated with or without simvastatin. Cells were then replaced with serum-free medium containing labeling media for 1 h. Labeling medium consisted of bodipy cholesterol (Avanti Polar Lipids), MCD, HEPES (Sigma-Aldrich; Merck KGaA), and egg phosphatidylcholine (Avanti Polar Lipids) and prepared as previously described (28 (link)). The final concentrations of bodipy cholesterol, egg phosphatidylcholine, and MCD in the labeling medium were 0.025 mM, 0.1 mM, and 10 mM, respectively. Cells were washed with HAM's F12-HEPES and incubated with serum-free medium for 18 h. ApoA-1 and HDL (both from Lee Biosolutions) were added for 6 h at 100 µg/ml and 70 µg/ml, respectively. Cells were dissolved with 1% cholic acid (Sigma-Aldrich; Merck KGaA) in 1N NaOH for 4 h with rocking while the supernatant was collected and centrifuged at 10,000 ×g for 5 min. The cholesterol fluorescence intensity value was recorded using a microplate reader (excitation at 482 nm and emission at 515 nm). The % cholesterol efflux = cholesterol efflux intensity/(intracellular cholesterol + cholesterol efflux) ×100%.
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!