Powder form of 3-sn-PA (Sigma Aldrich), Brain PS (Avanti Polar Lipids), Brain PI-(4)-P (Avanti Polar Lipids) was dissolved in chloroform in a clean glass vial and evaporated in a desiccator overnight at RT. Thin film of dried lipid obtained after drying was dissolved in 20 mM Tris pH 7.5 and 150 mM NaCl and stored in −20 °C for future use.
Brain ps
Manufactured by Avanti Polar Lipids
Brain PS is a phosphatidylserine (PS) lipid extract derived from bovine brain tissue. It is a naturally occurring phospholipid that plays a crucial role in various cellular processes. Brain PS provides the essential building blocks for the maintenance and function of neuronal membranes.
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Lab products found in correlation
3 sn pa, by Merck Group (2 mentions)
Malachite green, by Merck Group (2 mentions)
Ammonium molybdate, by Merck Group (2 mentions)
Egg pc, by Merck Group (1 mentions)
L α phosphatidylethanolamine, by Merck Group (1 mentions)
Cardiolipin, by Avanti Polar Lipids (1 mentions)
Biotinylated bovine serum albumin, by Thermo Fisher Scientific (1 mentions)
Oregon green 488, by Thermo Fisher Scientific (1 mentions)
Calcein, by Merck Group (1 mentions)
Dio and did, by Thermo Fisher Scientific (1 mentions)
Texas red maleimide, by Thermo Fisher Scientific (1 mentions)
Sulforhodamine b, by Merck Group (1 mentions)
Neutravidin, by Thermo Fisher Scientific (1 mentions)
Nbd pe, by Avanti Polar Lipids (1 mentions)
18 1 biotinyl cap pe, by Avanti Polar Lipids (1 mentions)
Brain pc, by Avanti Polar Lipids (1 mentions)
Anti lamp1, by Abcam (1 mentions)
Folch fraction 1, by Merck Group (1 mentions)
Anti β tubulin aa2, by Merck Group (1 mentions)
Anti kif5b, by Abcam (1 mentions)
7 protocols using brain ps
1
Lipid Film Reconstitution in Buffer
2
Artificial Lipid Bilayer Membrane Fabrication
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Powder form of Egg-PC (Sigma Aldrich), L-α-phosphatidylethanolamine (Sigma Aldrich), 16:0 Cardiolipin (Avanti Polar Lipids), 3-sn-PA (Sigma Aldrich), Brain PS (Avanti Polar Lipids) was dissolved in chloroform. Next, 270 μM PC, 145 μM phosphatidylethanolamine, 65 μM PIP4, 5 μM CL, 5 μM PA, 10 μM PS were mixed and kept for overnight evaporation. The thin lipid film obtained the following day after removal of residual chloroform was dissolved in 500 μl of 20 mM Tris pH 7.5 and 150 mM NaCl with gentle pipetting. The solution was next incubated at 55 °C for 30 min. The resultant colloidal heated solution was subjected to extrusion as mentioned above. Finally, the model membrane liposomes comprising a mixture of PC, phosphatidylethanolamine, PIP4, CL, PA, and PS were used for downstream experiments and the remaining was stored at 4 °C for not more than 3 days.
3
Lipid Labeling and Tracing Reagents
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Oregon Green 488 and Texas Red (TR)-coupled DHPE were purchased from Invitrogen (Waltham, MA). Lipophilic tracers—DiO and DiD, Texas Red maleimide (for protein labeling), NeutrAvidin, and biotinylated bovine serum albumin—were from Thermo Fisher Scientific (Waltham, MA). Calcein, sulforhodamine B, and other chemicals were from Sigma-Aldrich (St. Louis, MO). All other lipids were purchased from Avanti Polar Lipids (Alabaster, AL): brain-PC (L-α-phosphatidylcholine (Brain, Porcine)), brain-PE (L-α-phosphatidylethanolamine (Brain, Porcine)), brain-PS (L-α-phosphatidylserine (Brain, Porcine)), cholesterol (ovine wool), 18:1 Biotinyl Cap PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)), 18:1 NBD-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)), and 18:1 Liss Rho-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)).
4
Cellular Phosphatidylserine Quantification
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Total cellular lipids from indicated cells were extracted by chloroform/methanol extraction. TLC was performed as previously described [55 (link)]. Equal amounts of lipids were loaded onto a TLC plate based on protein quantification from individual cell lines and lipids were separated by TLC. Brain PS (Avanti Polar Lipids) and SM (Matreya LLC) were run as molecular standards. Bands corresponding to Brain PS were scraped and subjected to phosphorus extraction by acidic digestion. The liberated phosphorus was estimated by allowing a complex formation with ammonium molybdate (Sigma) and malachite green (Sigma) and by measuring the absorption at 660 nm [56 (link)]. Phosphorus was quantified using a standard curve obtained from phosphorus liberated from known concentrations of Brain PS run on TLC plate. Cellular PS was expressed as the ratio of phosphorus obtained from PS and phosphorus obtained from total phospholipids. Additionally, PS was estimated by acquiring TLC band intensities of PS and sphingomyelin (SM), using Image Studio Lite software; variations in PS are shown as a ratio of PS to SM bands.
5
Endosomal Trafficking Lipid Composition
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DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), bovine liver phosphatidylinositol (PI), brain PS, and egg PA were purchased from Avanti Polar Lipids. PI 3-phosphate diC16 [PI(3)P], PI 4-phosphate diC16 [PI(4)P], and PI 5-phosphate diC16 [PI(5)P] were obtained from Echelon Biosciences. Folch fraction I and egg PC were purchased from Sigma-Aldrich. YM201636 PIKfyve inhibitor was purchased from Cayman Chemical. A cell fractionation kit was obtained from Cell Signaling Technology. Nontargeting control siRNA was obtained from Horizon Discovery, and human KLC1/2 siRNAs were from Santa Cruz Biotechnology. The following primary antibodies were used: horseradish peroxidase (HRP)–conjugated anti-His6 (71841, Novagen), anti-GFP for immunoblotting (3E1, Roche), anti-actin (AC-74, Sigma-Aldrich), anti–β-tubulin (AA2, Sigma-Aldrich), anti–histone H3 (Abcam), anti-giantin (PRB-114C, Covance), anti-HA (HA-7, Sigma-Aldrich), anti-KLC1 ([EPR12441(B)], Abcam), anti-KLC2 (Abcam), anti-KIF5B (Abcam), anti-LAMP1 (lysosome-associated membrane protein 1) (D2D11, Cell Signaling Technology), anti-Rab7 (D95F2, Cell Signaling Technology), and anti-Rab6 (D37C7, Cell Signaling Technology). Alexa 568– and Alexa 633–conjugated anti-mouse or anti-rabbit secondary antibodies were from Thermo Fisher Scientific.
6
Quantification of Cellular Phospholipids
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Total cellular lipids were extracted by chloroform/methanol extraction. TLC was performed as previously described [14 (link)]. Lipids were loaded onto a TLC plate based on protein quantification. Brain PS (Avanti Polar Lipids) and sphingomyelin (Matreya LLC) were run as molecular standards. Bands corresponding to PS were scraped and subjected to phosphorus extraction by acidic digestion. The liberated phosphorus was estimated by allowing a complex formation with ammonium molybdate (Sigma) and malachite green (Sigma) and by measuring the absorption at 660 nm [54 (link)]. Phosphorus was quantified using a standard curve obtained from phosphorus liberated from known concentrations of Brain PS run on TLC plate. Cellular PS was expressed as the ratio of phosphorus obtained from PS and phosphorus obtained from total phospholipids. Additionally, PS was estimated by acquiring TLC band intensities of PS and sphingomyelin, using Image Studio Lite software; variations in PS are shown as a ratio of PS to sphingomyelin bands.
7
Phospholipid Labeling and Antibody Detection
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Natural and synthetic phospholipids, including POPC, POPS, Egg-PC, Brain-PS, Brain-PI(4,5)P2, and fluorescent TopFluor-TMR-PI(4,5)P2 and TopFluor-TMR-PS, are from Avanti Polar Lipids, Inc Oregon green 488-DHPE and Alexa Fluor 647 Maleimide labeling kit are from Invitrogen. Atto647N-DOPE was from Sigma. Monoclonal mouse anti-clathrin heavy chain (dilution 1:1000; Cat# 610499) was from BD Biosciences, and polyclonal rabbit anti-FCHo2 (dilution 1:1000; Cat# NBP2-32694) was from Novusbio.
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