Pellets containing a mix of embryos, larvae and adults were prepared by collecting the animals (grown on NGM agar plates with E. coli OP50 bacteria) in M9 buffer and washing thrice before removing all of the buffer and snap-freezing in liquid nitrogen. Extracts were prepared by grinding the pellet with a mortar and pestle in the presence of liquid nitrogen and dissolving in Lysis Buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% Triton X-100, 5% glycerol (w/vol), 5 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001), 1% P8340 (Sigma)). Extracts were cleared by centrifugation at 16100 x g for 5 minutes at 4°C. EEF-1A protein was enriched from the C. elegans extracts using a method previously employed for enrichment of eEF1A1 present in human cell extracts [21 (link)]. Extracts were loaded on Pierce Strong Cation Exchange (S) Spin Columns (Thermo Fisher Scientific, 90008) pre-equilibrated in Lysis Buffer, followed by extensive washing of the columns with Washing Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5% glycerol (w/vol), 5 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001), 1% P8340 (Sigma)). The retained material, including EEF-1A, was eluted from the columns with Elution Buffer (50 mM Tris-HCl pH 7.4, 400 mM NaCl, 5% glycerol (w/vol), 5 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001), 1% P8340 (Sigma)).
P8340
Manufactured by Merck Group
Sourced in United States, Germany, China, Sao Tome and Principe, Japan, United Kingdom, Australia, Spain, Switzerland
The P8340 is a laboratory centrifuge device manufactured by Merck Group. It is designed for general laboratory use, providing a reliable and efficient means of separating materials of different densities through centrifugal force.
Automatically generated - may contain errors
Lab products found in correlation
P5726, by Merck Group (86 mentions)
P0044, by Merck Group (63 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (36 mentions)
Pvdf membrane, by Merck Group (20 mentions)
P2850, by Merck Group (17 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Image lab software, by Bio-Rad (10 mentions)
Bca assay, by Thermo Fisher Scientific (10 mentions)
Protease inhibitor cocktail, by Merck Group (10 mentions)
Bca protein assay, by Thermo Fisher Scientific (10 mentions)
Imagequant las 4000, by GE Healthcare (9 mentions)
Ripa buffer, by Merck Group (9 mentions)
Gapdh, by Santa Cruz Biotechnology (8 mentions)
β actin, by Merck Group (8 mentions)
Odyssey imaging system, by LI COR (7 mentions)
Protease inhibitor, by Merck Group (7 mentions)
Odyssey blocking buffer, by LI COR (6 mentions)
R0278, by Merck Group (6 mentions)
P7626, by Merck Group (6 mentions)
Chemidoc mp imaging system, by Bio-Rad (6 mentions)
471 protocols using p8340
1
Enrichment and Purification of EEF-1A Protein from C. elegans
2
Protein Extraction and Quantification
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Cells were harvested and lysed in 1 × radioimmune precipitation assay buffer (1% NP-40, 50 mmol/L Tris-HCl/pH 7.4, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid (EDTA) containing protease inhibitor mixture (P8340; Sigma; 1:1,000 dilution), and 0.625 mg/mL N-ethylmaleimide (E3876; Sigma). After incubation on ice for 20 minutes, the lysates were cleared by centrifugation at 1,000 × g for 10 minutes at 4°C and the protein concentrations were determined using a BCA assay (Thermo Scientific, Waltham, MA, USA).
Dissected mouse spinal cords were lysed in radioimmune precipitation assay buffer supplemented with protease inhibitor mixture (P8340; Sigma; 1:1,000 dilution), 0.2 mmol/L phenylmethylsulfonyl fluoride, and 0.625 mg/mL N-ethylmaleimide. Lysates were cleared by centrifugation at 1,000 × g for 10 minutes at 4°C, and the protein concentration was determined by BCA assay.
Proteins were denatured by boiling in 6 × SDS sample buffer for 5 minutes at 95°C to prepare for SDS-polyacrylamide gel electrophoresis (PAGE).
Dissected mouse spinal cords were lysed in radioimmune precipitation assay buffer supplemented with protease inhibitor mixture (P8340; Sigma; 1:1,000 dilution), 0.2 mmol/L phenylmethylsulfonyl fluoride, and 0.625 mg/mL N-ethylmaleimide. Lysates were cleared by centrifugation at 1,000 × g for 10 minutes at 4°C, and the protein concentration was determined by BCA assay.
Proteins were denatured by boiling in 6 × SDS sample buffer for 5 minutes at 95°C to prepare for SDS-polyacrylamide gel electrophoresis (PAGE).
3
Cellular Fractionation for Protein Extraction
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MDA-MB-468 and SW527 cells were digested, washed twice with cold PBS, and harvested by centrifuging for 5 min at 250 g (4 ℃). The cells were incubated in 150 μl of Cytoplasm Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP40, and 1% protease inhibitor cocktail (P-8340, Sigma, St. Louis, MO)) for 10 min at 4℃. The supernatant obtained by centrifuging is the cytoplasmic protein fraction. The pellet was washed once with the Cytoplasm Lysis Buffer and extracted using 60 μl of Nuclear Lysis Buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS, 1% (P-8340, Sigma, St. Louis, MO)) with a 30 min vortex.
4
Nuclear Protein Extraction Protocol
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For nuclear protein extraction cells were trypsinized (0.025% trypsin and 0.04% EDTA, SIGMA 25300-054) at 37 °C, pelleted at 300 rcf and washed twice with cold PBS. During each wash cells were pelleted at 300rcf for 5 min at 4 °C. Cells were incubated in hypotonic buffer (10 mM Tris-HCl pH 7.8, 5 mM KCl, 2 mM MgCl2 DTT 1 mM) containing protease inhibitors (SIGMA P8340) for 10 min at 4 °C. Cells were pelleted at 300 rcf for 5 min at 4 °C and plasma membrane lysis was performed in 0,25% NP-40 hypotonic buffer on ice for 15 min. Nuclei were pelleted at 300 rcf for 15 min at 4 °C and washed twice in hypotonic buffer. Isolated nuclei were incubated in RIPA buffer (150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl, pH 8.0) containing protease (SIGMA P8340) and phosphatase inhibitors (SIGMA P2850). Insoluble material was pelleted by centrifugation at 16,000 rcf for 30 min at 4 °C. Protein concentrations were determined using the Bradford assay (Bio-Rad 500-0006). Western blot was performed as specified in the apposite section with the antibodies indicated in Table S6 .
5
Integrin and Cadherin Expression Analysis
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Cells were trypsinized, washed and lysed in RIPA Buffer (25mMTris-HCL pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) with protease (P8340, Sigma Aldrich) and phosphate cocktails (P8340, Sigma Aldrich). The lysates were separated by 4–20% precast polyacrylamide gel (BioRad) and proteins transferred to PVDF membranes (BioRad), blocked for 1 hour with 5% defatted milk in a PBS/0.5% Tween 20 at room temperature. Samples were immunoassayed for αv, β3, α5, β1, E-cadherin, N-cadherin and α-Tubulin (Cell Signaling, USA) using Pierce ECL Western Blotting Substrate (Thermo Scientific).
6
Fractionation of HUVEC Proteins
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HUVEC challenged with or without intermittent hyperglycemia, were harvested and washed with PBS. Upon centrifugation, the cell pellet was re-suspended in ice cold PBS containing 0.1% Nonidet P-40 (NP-40) and 1% protease inhibitors (#P8340, Sigma-Aldrich, MI, USA). The nuclear fraction was pelleted down by centrifugation for 10 min at 10,000 rpm, while the supernatant was collected as the membrane and cytoplasmic fraction. Furthermore, the nuclear lysate was obtained by resuspending the nuclear pellet in PBS containing 0.1% Nonidet P-40 (NP-40) and 1% protease inhibitor (#P8340, Sigma). Both the nuclear and cellular fractions were sonicated for 10 s (×2) and then separated by SDS-PAGE and immunoblotted.
7
Subcellular Fractionation and Protein Quantification
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HeLa and T47D cells were washed twice with PBS and lysed by sonication in RIPA lysis buffer (50 mM Tris-HCl, pH 7.5; 1% NP-40, 2 mM Na3VO4, 150 mM NaCl, 20 mM Na4P2O7, 100 mM NaF), supplemented with a complete protease inhibitor cocktail (P8340, Sigma) and phosphatase inhibitor cocktail (P5726, Sigma).
To detect nuclear NICD by WB, HeLa and T47D cells were trypsinized, washed twice with cold PBS, and incubated for 30′ in hypotonic buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1 mM Na-EDTA, 1 mM Na-EGTA, 1.5 mM MgCl2, 2 mM dithiothreitol (DTT)) supplemented with a complete protease inhibitor cocktail (P8340, Sigma) and a phosphatase inhibitor cocktail (P5726, Sigma). Afterward, cells were lysed by repeated passage through a 22G needle and centrifuged at 10000 g for 10′ at 4°C. The pellet was considered as a nuclei-enriched fraction and sonicated using complete RIPA buffer. This nuclei-enriched fraction was certified by WB due to the presence of hnRNP C1/C2 only in this fraction compared to the supernatant (cytoplasmic fraction, data not shown).
Total protein quantification was performed with Bio-Rad Protein Assay Dye Reagent concentrate.
To detect nuclear NICD by WB, HeLa and T47D cells were trypsinized, washed twice with cold PBS, and incubated for 30′ in hypotonic buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1 mM Na-EDTA, 1 mM Na-EGTA, 1.5 mM MgCl2, 2 mM dithiothreitol (DTT)) supplemented with a complete protease inhibitor cocktail (P8340, Sigma) and a phosphatase inhibitor cocktail (P5726, Sigma). Afterward, cells were lysed by repeated passage through a 22G needle and centrifuged at 10000 g for 10′ at 4°C. The pellet was considered as a nuclei-enriched fraction and sonicated using complete RIPA buffer. This nuclei-enriched fraction was certified by WB due to the presence of hnRNP C1/C2 only in this fraction compared to the supernatant (cytoplasmic fraction, data not shown).
Total protein quantification was performed with Bio-Rad Protein Assay Dye Reagent concentrate.
8
Mitochondrial Membrane Fractionation
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Tissue samples were rapidly homogenized in 20 mM Tris-HCl (pH 7.4), 0.32 M sucrose, 1 mM ethylenediaminetetraacetic acid, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 0.5 mM methyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraaceticacid and a cocktail of protease inhibitors (Sigma, P8340), and centrifuged at 1000 g for 10 min. One third of the supernatant was centrifuged at 15,000 g for 20 min. The resulting pellet was washed once and dissolved in 5% sodium dodecylsulphate (SDS) and is referred to as the P2 mitochondrial-membrane fraction. The rest of the supernatant was centrifuged also at 15,000 g for 20 min and the resulting pellet solubilized in RIPA buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% Nonidet P-40, 2.5 mg/ml sodium deoxycholate, 1 mM Na3VO4) and a cocktail of protease inhibitors (Sigma, P8340) for two hours at 4°C with periodic stirring.
9
Nuclear and Cytoplasmic Protein Fractionation
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Cells were washed with PBS twice, scrape off and pelleted by centrifuging at 4,500 g for 5 min. Cells were then swelled by adding 5 volume of lysis buffer (10 mM HEPES, pH 7.9, with 1.5 mM MgCl2, 10 mM KCl, 1mM DTT and protease inhibitor, sigma, P8340) and homogenized. After centrifugation at 10,000 g for 15 min, the supernatant was collected as a cytoplasmic fraction. The crude nuclei pellet was resuspended in 2/3 volume extraction buffer (20 mM HEPES, pH 7.9, with 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 25% (v/v) Glycerol, 1mM DTT and protease inhibitor sigma P8340) and homogenized with a tissue homogenizer. After centrifuging at 20,000 g for 5 min, the supernatant was collected as a nuclear fraction.
10
Fractionation of Cellular Proteins
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Fractionation of cytoplasmatic and nuclear proteins was performed using three buffers: HB-Buffer (10 mM Tris-HCl, pH 8, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM 2-mercaptoethanol, protease inhibitor [P8340, Merck; 4 µL per 5 mL]); Lysis-Buffer: HB-Buffer + 0.4% NP-40); Buffer C (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, protease inhibitor [P8340, Merck; 4 µL per 1 mL]). 1–5 × 106 cells were harvested, pelleted at 200× g for 5 min and washed at RT. The cell pellet was resuspended in 350 µL HB-Buffer and centrifuged at 200× g for 1 min at 4 °C. The supernatant was removed and the pellet was resuspended in 100 µL Lysis-Buffer followed by incubation for 15 min at 4 °C. The supernatant containing the cytosolic fraction was harvested by centrifugation at 15,000× g for 5 min at 4 °C. The pellet was washed twice with 75 µL Lysis-Buffer, resuspended in 60 µL Buffer C and incubated for 15 min at 4 °C. Following centrifugation at 15,000× g for 5 min at 4 °C the supernatant containing the nuclear fraction was harvested. Protein concentrations were then determined using the Protein Assay Kit I (Bio Rad, 5000001).
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