Myomerger proteins were incubated with DOPC:DiI (98:2) or DOPC:DOPS:DiI (68:30:2) at 1:100 (protein:lipid) for 10 min at room temperature. Twenty-four microliters of 50% (weight/volume; wt/vol) Ficoll (Sigma-Aldrich, F4375) in liposomal buffer was added and thoroughly mixed with the protein-liposome sample in 230-µL thick-walled polycarbonate tubes (Beckman Coulter). One hundred microliters of 10% (wt/vol) Ficoll was overlaid onto the mixture and 10 µL of liposomal buffer was added as the top layer. Samples were spun at 200,000 × g for 90 min at 4 °C in a TLA-100 fixed-angle rotor (Beckman Coulter) in a MAX-XP ultracentrifuge (Beckman Coulter). After centrifugation, the vesicles floated to the 0/10% Ficoll interface, and 30-µL aliquots were taken from the top and the bottom and analyzed with SDS-PAGE and Western blotting with Myomerger antibodies.
Tla 100 fixed angle rotor
Manufactured by Beckman Coulter
Sourced in United States
The TLA-100 fixed-angle rotor is a laboratory equipment designed for high-speed centrifugation. It is primarily used to separate and isolate various components within a sample. The rotor's fixed-angle configuration allows for efficient separation of particles or molecules based on their size and density.
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4 protocols using tla 100 fixed angle rotor
1
Myomerger Protein-Liposome Flotation Assay
2
Amyloid-beta Aggregation Kinetics
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Aβ42 monomers (0.25 μg/μl assay buffer A) were placed on ice, or incubated for 30 min or 60 min (2 x 100 μL/condition) at 1000 rpm/37°C to form multimers. The multimer samples were transferred to thick-wall polycarbonate tubes (Beckman Coulter) and subjected to centrifugation at 20,000 rpm (15,456 × g) for 30 min (Optima Max-TL Tabletop Ultracentrifuge (Beckman Coulter); TLA-100 fixed-angle rotor (Beckman Coulter)). Supernatant (100 μL) was removed carefully from the 30 min sample so as to not disturb the pellet, and placed on ice. For the 60 min sample, supernatant was removed by pipet and the pellet was resuspended in 200μL assay buffer A. Following a second spin (20,000 rpm × 30 min), all supernatant was removed carefully, and the pellet was resuspended in 40 μL assay buffer A. The protein concentration of each sample was determined by the bicinchoninic acid (BCA) method according to the manufacturer’s directions (Pierce).
3
Lipoprotein Fractions Purification by Ultracentrifugation
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Lipoprotein fractions were purified by isopycnic salt gradient ultracentrifugation (Figure 5 ) [3 (link),4 (link)]. Briefly, 0.9 mL of plasma sample, brought to d = 1.3 g/mL with solid NaBr (472.2 mg NaBr/mL plasma), were gently overlaid with 2.1 mL of a d = 1.006 g/mL solution (0.6% NaCl) (Figure 5 a), and centrifuged at 541,000× g for 3 h at 4 °C in a TL-100 series ultracentrifuge equipped with a TLA-100 fixed-angle rotor (Beckman Coulter, Indianapolis, IN, USA) (Figure 5 b). Afterwards, VLDL (d = 1.006–1.063 g/mL), LDL (d = 1.063–1.19 g/mL) and HDL (d = 1.19–1.21 g/mL) fractions were collected and further purified by a second centrifugation step, performed at 541,000× g for 2 h in saline solutions at density 1.006, 1.063, and 1.21 g/mL (Figure 5 c), respectively, followed by desalting and concentration using Amicon Ultra-0.5 mL centrifugal filter units (10 KDa MWCO, Merck-Millipore, Darmstadt, Germany). The degree of purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as previously described [3 (link)].
4
Lipin 1 Membrane Binding Assay
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For the full-length lipin 1 and ΔM-Lip lipin 1 proteins, 20 µL of LUV liposomes in 50 mM Tris, pH 7.5, 100 mM NaCl, 10 mM 2-mercaptoethanol buffer were mixed with 20 µL of proteins to give a final concentration of 1.0 mM liposomes and 1.0 µM protein. For the M-Lip and M-Lipxtal proteins, 50 μL of LUV liposomes in pH 7.4 PBS buffer were mixed with 50 µL of proteins in PBS buffer, giving a final concentration of 1 mM liposomes and 50 µM protein. Reaction mixtures were incubated for 30 minutes and centrifuged at 100,000 × g at 4 °C for 1 h using a TLA100 fixed angle rotor (Beckman). The supernatant fraction was carefully removed, and the protein content of the pellet and supernatant fractions were analyzed by SDS-PAGE. All binding assays were performed at least three times and SDS-PAGE gel bands were quantified using ImageJ43 .
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