Fibril-treated primary neuron lysates were prepared from a petri dish as previously described for velocity and density gradients. For extraction of insoluble proteins, 500 μl of solubilized lysates were mixed 1:1 with SB 40% (w/v) sucrose, without sarkosyl, MgCl2, and Benzonase, in 1-ml thickwall polycarbonate ultracentrifuge tubes (Beckman Coulter) and centrifuged at 250,000g for 1 hour at room temperature with a TLA 120.2 rotor using an Optima XP benchtop ultracentrifuge (Beckman Coulter). Supernatants were collected by pipetting. Pellets were resuspended in 50 μl of PBS, and total protein concentration was determined by bicinchoninic acid assay (Pierce) before equalization and denaturation for 5 min at 100°C in Laemmli buffer.
Thickwall polycarbonate ultracentrifuge tubes
Manufactured by Beckman Coulter
Thickwall polycarbonate ultracentrifuge tubes are designed for use in high-speed centrifugation applications. They are constructed with a thick polycarbonate material that provides enhanced durability and resistance to breakage. These tubes are suitable for a variety of ultracentrifugation procedures, where their structural integrity and compatibility with the instrumentation are critical factors.
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Lab products found in correlation
2 protocols using thickwall polycarbonate ultracentrifuge tubes
1
Extraction of Insoluble Neuronal Proteins
2
HDL Isolation from Serum Samples
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HDL was isolated from 200 μl aliquots of serum as follows: Serum samples were added to a mixture containing 1 part 500iu/ml heparin (Mucosal, Fresenius) and 2 parts 1.12 (mol/L) manganese chloride solution. Samples were centrifuged at 10 000 g for 1 h at 4 °C. The supernatant was dialysed against phosphate buffered saline (PBS, pH 7.4) in Spectra/Por 2 RC membrane (12 000–14 000 kDa) (GIC Scientific, 132676) and 200 μl aliquots were then dissolved in sodium bromide (275.5 mg/ml of supernatant), transferred to thick-wall polycarbonate ultracentrifuge tubes (Beckman, 343775) and centrifuged at 223 000 g for 20 h at 4 °C and the upper 70 μl layer was extracted. Purity was confirmed using 12.5 % reducing SDS-polyacrylamide gel electrophoresis (PAGE) stained with Coomassie Blue. The protein concentration of HDL was determined by the modified Lowry method [25 (link)]. All samples were analysed in duplicate.
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