Lipids were purchased as L-α-Phosphatidylcholine (PC), L-α-Phosphatidylethanolamine (PE), L-α-Phosphatidylinositol (PI), L-α-Phosphatidylserine (PS) and Cardiolipin (CL) from Avanti Polar Lipids (AL, USA). The lipid mixture of 45:20:15:5:15 mol% PC:PE:PI:PS:CL in CHCl3, closely resembling inner mitochondrial lipid composition (van Meer et al., 2008 (link)), was dried with a nitrogen stream and resuspended in 100 mM KCl, 10 mM MOPS-Tris, pH 7.0. Lipid suspension was thoroughly vortexed, subjected to seven freeze-thaw cycles and extruded through 200 nm membranes (Whatman plc, UK) to ensure unilamellarity and defined size distribution. Both liposomes and protein in urea were incubated with the mild detergent MEGA-9 (Glycon, DE) above CMC at 80 mM first separately then combined, at room temperature. Subsequently the liposome-protein-detergent mixture was subjected to dialysis against 5 L of liposome buffer to remove both urea and MEGA-9. Incorporation success was monitored by Histodenz flotation assay and sodium carbonate extractions as described elsewhere (Barbot et al., 2015 (link)).
L α phosphatidylserine
Manufactured by Avanti Polar Lipids
Sourced in United States, United Kingdom
L-α-phosphatidylserine is a phospholipid compound that is a natural component of cell membranes. It plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
L α phosphatidylcholine, by Avanti Polar Lipids (10 mentions)
L α phosphatidylethanolamine, by Avanti Polar Lipids (7 mentions)
L α phosphatidylinositol, by Avanti Polar Lipids (4 mentions)
Cardiolipin, by Avanti Polar Lipids (3 mentions)
Whatman, by Cytiva (2 mentions)
Fviia, by Novo Nordisk (2 mentions)
Formvar carbon coated copper grid, by Agar Scientific (2 mentions)
L α phosphatidylinositol 4 5 bisphosphate pip2, by Avanti Polar Lipids (2 mentions)
L α phosphatidylcholin, by Avanti Polar Lipids (2 mentions)
Jem 1011 transmission electron microscope, by JEOL (2 mentions)
Orius 1000 ccs detector, by Ametek (2 mentions)
1 2 dimyristoyl sn glycero 3 phosphocholine dmpc, by Avanti Polar Lipids (1 mentions)
Polycarbonate filter, by Cytiva (1 mentions)
Bio beads sm 2 resin, by Bio-Rad (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Anti cd61 percp, by BD (1 mentions)
Carbonyl cyanide m chlorophenyl hydrazone, by Merck Group (1 mentions)
Sodium azide nan3, by Merck Group (1 mentions)
Tetraethylammonium chloride tea, by Merck Group (1 mentions)
Ergosterol, by Merck Group (1 mentions)
17 protocols using l α phosphatidylserine
1
Reconstitution of Membrane Proteins in Liposomes
2
Formation of oAβ-lipoprotein Complexes
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L-α-phosphatidylserine (PS) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Avanti Polar Lipids as powder. PS and DMPC were dissolved in chloroform at 10 mg/mL and mixed at 1:4. Chloroform was evaporated under vacuum and formed a thin layer containing the mixture of PS and DMPC. DPBS was added to re-hydrate the lipid mixture to 5 mg/mL, and the liposomes were formed by sonication on ice until the solution becomes translucent. To prepare oAβ-lipoprotein complexes, PS/DMPC liposomes and APOE3 were mixed at final concentrations of 1 mg/mL for PS/DMPC liposomes and 0.25 mg/mL for APOE3. The mixture was incubated at 18 °C 15 min and 30 °C 15 min for 3 cycles [28 (link)]. Then FAM-labeled oAβ was added into the lapidated APOE at a final concentration of 1 μM and incubated at room temperature for 1 hr.
3
Reconstitution of Membrane Proteins in Lipid Vesicles
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l -α-Phosphatidylcholine (PC), l -α-phosphatidylethanolamine (PE), l -α-phosphatidylinositol (PI), l -α-phosphatidylserine (PS), cardiolipin (CL), and 1,2-dioleoyi-s-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids, Inc. 20% DOPE/80% PC, 20% DOPE/65% PC/15% CL, 60% PC/20% PE/15% PI/5% PS, and 45% PC/20% PE/15% PI/5% PS/15% CL lipid mixtures were dried under continuous N2 flow to form thin lipid films and additionally dried in a desiccator for at least 3 h. Lipid films were hydrated in 150 mM NaCl and 20 mM Hepes buffer, pH 7.4. LUVs were formed by repeated freeze–thaw cycles, followed by extrusion through a 100-nm diameter polycarbonate filter (Whatman). Protein was added to presolubilized liposomesin 0.1% DDM LUVs with a molar protein/lipid ratio of 1:450 in 350 µl incorporation mixture. Proteoliposomes were formed by DDM depletion by using detergent-adsorbent Bio-Beads SM-2 Resin (Bio-Rad Laboratories).
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4
Recombinant TF Relipidation in Vesicles
5
Coagulation Pathway Protein Quantification
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The following materials were obtained from the sources shown in parentheses: thromboplastin (TF), kaolin (Renam, Moscow, Russia), fVIIa (Novo Nordisk, Bagsvaerd, Denmark) fVa, fIXa, fXIa, fV, fIX, Anti-Human Factor XI antibody (Haematologic Technologies Inc., Vermont, USA), fII (Enzyme Research Laboratories Inc, South Bend, IN, USA), fVIII (Hemophil M, Baxter, Moscow, Russia), L-α-phosphatidylserine (Brain, Porcine) (PS), L-α-phosphatidylcholine (Egg, Chicken) (PC) (Avanti Polar Lipids, Alabaster, Alabama, USA), specific fXIIa inhibitor corn trypsin inhibitor (CTI) (Institute of Protein Research, Russian Academy of Sciences, Pushchino, Russia), D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK), (Calbiochem, Emd Biosciences Inc, San Diego, CA, USA), mouse anti-human CD61 labeled with peridinin-chlorophyll-protein complex (anti-CD61-PerCP), annexin V-FITC (BD Biosciences, San Jose, CA, USA), chromogenic substrate for fXIIa, fXIa and kallikrein S2302 (CHROMOGENIX, Instrumentation Laboratory company, Bedford, MA, USA), fluorogenic substrate for thrombin Z-Gly-Gly-Arg-AMC·HCI (Bachem Americas, Inc., Torrance, CA, USA). Nitrophorin 2 was prepared as described [16] . All other reagents were obtained from Sigma-Aldrich (St. Louis, Missouri, USA).
6
Fungal Growth Assay Protocols
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Sabouraud’s Dextrose Agar was purchased from Acumedia (Michigan, MI, USA). Peptides were purchased from EZBiolabs Inc., (Carmel, IN, USA). Lipids such as L-α-phophatidylcholine (PC), L-α-phostidylethanolamine (PE), L-α-phosphatidylserine (PS) and L-α-phosphatidylinositol (PI) were bought from Avanti Polar Lipids Inc. (AL, USA). Ergosterol, sodium azide (NaN3), Carbonyl cyanide m-chloro phenylhydrazone (CCCP), 4-aminopyridine (4-AP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), Gadolinium III Chloride, tetra ethyl ammonium chloride (TEA) and 3′,3′-dipropylthiadicarbocyanine (diS-C3-5) dye were purchased from Sigma-Aldrich Corp. (MO, USA). ATP bioluminescence kit was obtained from Molecular Probes Inc (OR, USA). Amphotericin B and Natamycin were obtained in powder form from Sigma-Aldrich (S) Pte Ltd (Singapore).
7
Lipid Standards Analysis Protocol
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Lipid standards were purchased
from Avanti Polar Lipids [l -α-phosphatidylethanolamine
(Brain, Porcine) (PE),l -α-phosphatidylserine (brain,
porcine) (sodium salt) (PS), and 1′,3′-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol (sodium salt) (18:1 cardiolipin)
(CL)] and were diluted in CHCl3 whilel -α-phosphatidylcholine
(brain, porcine) (PC) was diluted in MeOH. Glucosylsphingosine (GlcSph)
(Sigma-Aldrich) was diluted in MeOH. All standards were analyzed using
a slightly varied analytical method to that of the patient samples;
the changes were as follows: a 5 min solvent vent was applied in addition
to a further 4 min high-temperature hold at the end of the GC temperature
gradient. The analytical method was 25 min in total. From a standard
solution of 100 μM total, a 20 μL portion was analyzed
in separate headspace vials. Lipid standards were measured in triplicate
alongside blank headspace vials and blank solvent analyses (both MeOH
and CHCl3) which were measured both prior to and after
lipid standard analysis. A comparative example of a lipid standard
(l -α-phosphatidylethanolamine (Brain, Porcine) (PE)),
blank headspace vial, and solvent-only analysis is reported as overlaid
chromatograms inFigure S1C .
from Avanti Polar Lipids [
(Brain, Porcine) (PE),
porcine) (sodium salt) (PS), and 1′,3′-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol (sodium salt) (18:1 cardiolipin)
(CL)] and were diluted in CHCl3 while
(brain, porcine) (PC) was diluted in MeOH. Glucosylsphingosine (GlcSph)
(Sigma-Aldrich) was diluted in MeOH. All standards were analyzed using
a slightly varied analytical method to that of the patient samples;
the changes were as follows: a 5 min solvent vent was applied in addition
to a further 4 min high-temperature hold at the end of the GC temperature
gradient. The analytical method was 25 min in total. From a standard
solution of 100 μM total, a 20 μL portion was analyzed
in separate headspace vials. Lipid standards were measured in triplicate
alongside blank headspace vials and blank solvent analyses (both MeOH
and CHCl3) which were measured both prior to and after
lipid standard analysis. A comparative example of a lipid standard
(
blank headspace vial, and solvent-only analysis is reported as overlaid
chromatograms in
8
Liposome Floatation Assay for BECN1 Binding
9
Recombinant sTF (1–219) Protein Production
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Recombinant sTF (1–219) was derived from Escherichia coli (15 (link), 20 (link)) unless otherwise stated. FVIIa was from Novo Nordisk A/S. l -α-phosphatidylcholine (chicken egg) and l -α-phosphatidylserine (porcine brain) from Avanti Polar Lipids were used for the preparation of 10:90 PS:PC vesicles according to published procedures (47 (link)).
Unless otherwise stated, experiments were performed in HBS buffer (20 mM Hepes, 100 mM NaCl, pH 7.4) at room temperature and data were analyzed using GraphPad Prism.
Unless otherwise stated, experiments were performed in HBS buffer (20 mM Hepes, 100 mM NaCl, pH 7.4) at room temperature and data were analyzed using GraphPad Prism.
10
Polyphosphate Preparation and Characterization
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