To generate cell lysates, HEK293FTs were washed with cold PBS and lysed with ice‐cold radioimmunoprecipitation assay buffer (150 mm NaCl, 50 mm Tris‐HCl pH 8.0, 1% Triton X‐100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with protease inhibitor (Pierce/Thermo Fisher #A32953). After a 30 min incubation on ice, lysates were centrifuged at 14 000 × g for 20 min at 4 °C. Protein concentration for each sample was evaluated using a bicinchoninic acid (BCA) assay (Pierce/Thermo Fisher #23225). Samples were kept on ice until use or frozen at −80 °C for long term storage.
A32953
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A32953 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It serves as a core function in various laboratory settings, but a detailed description while maintaining an unbiased and factual approach cannot be provided. Additional information is not available.
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Lab products found in correlation
Protein assay reagent, by Bio-Rad (4 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Anti ha agarose beads a2095, by Merck Group (2 mentions)
Anti flag agarose beads a 2220, by Merck Group (2 mentions)
Orbitrap fusion, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1200 system, by Thermo Fisher Scientific (1 mentions)
A2220, by Merck Group (1 mentions)
A2095, by Merck Group (1 mentions)
Du 800 spectrophotometer, by Bio-Rad (1 mentions)
B15002, by Selleck Chemicals (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Sangon (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Las 4000, by Cytiva (1 mentions)
Anti prmt5 antibody, by Santa Cruz Biotechnology (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
18 protocols using a32953
1
Generating Cell Lysates from HEK293FT Cells
2
Cell Lysis and Protein Extraction
3
Quantitative Proteomic Analysis of Cell Lysates
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Cells were lysed with a lysis buffer [50 mM ammonium bicarbonate, 8 M urea, and protease inhibitor (A32953, Thermo Scientific)] under ultrasound in ice for 30 min. After centrifugation (14,000 rpm, 15 min, 4°C), 5 mM dithiothreitol (DTT) at 37°C for 1 h, 10 mM iodoacetamide at room temperature for 45 min and 50 mM ammonium bicarbonate to diluted urea to 1 M were performed in sequence. The protein solution was then digested by trypsin in a ratio of 1:50 at 37°C overnight. Peptides were separated and analyzed on an Easy-nLC 1,200 system coupled to an Orbitrap Fusion (Thermo Fisher Scientific). Raw data were processed and searched with MaxQuant 1.5.4.1 with an MS tolerance of 5 ppm and an MS/MS tolerance of 20 ppm. The UniProt human protein database (release 2016_07, 70,630 sequences) and the database for proteomics contaminants from MaxQuant were used for database searches. Student’s t test was used to determine the statistical significance of protein abundances between test and control samples. A p-value <0.05 was considered to be statistically significant. A second threshold based on a fold change of greater than 1.5-fold or less than 1.5-fold was chosen so as to focus the data analysis on a small set of proteins with the largest alterations in abundance.
4
Cell Lysis and Immunoprecipitation Protocol
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Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (A32953, Thermo Fisher) and phosphatase Inhibitors (B15002, Bimake). The protein concentrations of lysates were measured using the Beckman Coulter DU-800 spectrophotometer and the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, cell lysates containing 1 mg of total proteins were incubated with anti-Flag agarose (A2220, Sigma) or anti-HA Agarose (A2095, Sigma) for 4 hours at 4 °C. Precipitants were washed three times with EBC buffer and resolved by SDS-PAGE followed by immunoblot analysis with indicated antibodies.
5
Western Blot Analysis of Protein Lysates
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Cells were lysed in ice-cold RIPA (9,806, Cell Signaling) with PMSF (A100754, Sangon Biotech) and protease inhibitors (A32953, Thermo Scientific) for 20–30 min, followed by centrifugation, quantification, and denaturation. Twenty micrograms protein from each sample was separated to 8% SDS-PAGE gels, then transferred onto PVDF membranes and blocked with 5% non-fat milk. Membranes were sequentially incubated with primary antibodies and HRP-labeled secondary antibodies. The signals were detected by chemiluminescence using the ChemiDoc MP Imaging System (Bio-Rad). The bands’ intensity was measured by the GelPro Analyzer software.
6
Cytokine Profiling in Post-Surgical Mice
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Serum and the hippocampus tissues were collected at indicated time points after surgery in mice from the control, single anesthesia/surgery, and multiple anesthesia/surgery groups. The harvested brain tissues were homogenized on ice using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) with protease inhibitors (A32953, Thermo). After collection of the whole blood, it was left undisturbed at room temperature for approximately 30 min. Following centrifugation at 3000 ×g for 10 min at 4°C, the supernatant (serum) was collected. Levels of IL-1β, TNF-α, IL-6, and IL-10 in both serum and hippocampus tissue were measured using mouse ELISA kits (MultiSciences Biotechnology, Hangzhou, China).
7
Extraction of Soluble Aggregates from PD and DLB Brains
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Soluble aggregates were extracted from the brain tissues of PD and DLB donors (SI Appendix, Table S1 ), following the protocol as described in Hong et al. (76 (link)). The specimens were obtained from the London Neurodegenerative Diseases Brain Bank, Brains for Dementia Research, and the Multiple Sclerosis and Parkinson’s Tissue Bank. Frozen temporal cortical tissues (∼0.5 g) were diced and incubated with artificial cerebrospinal fluid (aCSF) (124 mM NaCl, 2.8 mM KCl, 1.25 mM NaH2PO4, and 26 mM NaHCO3, pH 7.4) supplemented with protease inhibitors (A32953; Thermo Fisher Scientific) for 30 min at 4 °C. Samples were centrifuged for 10 min at 2,000 × g, and the supernatant was collected for subsequent ultracentrifugation at 200,000 × g for 110 min at 4 °C in a SW41 Ti rotor. The upper 80% of the resulting supernatant was dialyzed against a 100-fold excess of fresh aCSF for 72 h, with buffer changed every 24 h. Protein concentrations of the dialyzed samples were determined by Bradford assay, aliquoted, and stored at -80 °C.
8
Uterine Protein Expression Analysis
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When comparing ISs and IISs, the uterus was removed and ISs and IISs were separated. The IS was then cut longitudinally and the embryo was carefully separated from the uterus under a dissecting scope. In all circumstances, the uterine segments contained myometrium and endometrium. Tissues were then snap frozen at after processing.
Uterine tissues were homogenized in radioimmunoprecipitation assay buffer supplemented with phosphatase and protease inhibitors (Sigma‐Aldrich; P0044‐1ML and Thermo Fisher; A32953, respectively), and the protein concentration was determined by BCA assay. Proteins (25 μg) were separated in a 10% SDS‐PAGE gel using constant voltage. Then, proteins were transferred onto polyvinylidene difluoride membranes and were blocked for 1 h with either 5% BSA in TBS‐T or 5% nonfat dry milk in TBS‐T depending on the primary antibody. The membranes were incubated in primary antibody (Table1 ) overnight at 4°C. The next day, the membranes were washed three times with TBS‐T and incubated in block in TBS‐T containing anti‐rabbit secondary antibody (Cell Signaling; 7074S) for 30 min. SuperSignal West Pick PLUS chemiluminescent substrate (Thermo Scientific; 34577) was used for developing signals and images were obtained using an ImageQuant LAS 4000 (GE Healthcare)
Uterine tissues were homogenized in radioimmunoprecipitation assay buffer supplemented with phosphatase and protease inhibitors (Sigma‐Aldrich; P0044‐1ML and Thermo Fisher; A32953, respectively), and the protein concentration was determined by BCA assay. Proteins (25 μg) were separated in a 10% SDS‐PAGE gel using constant voltage. Then, proteins were transferred onto polyvinylidene difluoride membranes and were blocked for 1 h with either 5% BSA in TBS‐T or 5% nonfat dry milk in TBS‐T depending on the primary antibody. The membranes were incubated in primary antibody (Table
9
Triinean Tricuspid Valve Isolation and Morphometric Analysis
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Immediately after tricuspid valve isolation, we cut open the valve leaflet complex by separating the leaflets at the posterior-septal commissure to enable valve unfolding. We floated the tricuspid valve, atrialis side up in 1xPBS and orthogonally photographed the tricuspid valve leaflets on a calibrated grid. Using these photographs, an experienced cardiac surgeon identified the commissural points at which leaflets were separated and, using custom MATLAB code, we calculated the anterior leaflet area, major cusp width, and major cusp height (Figure 1a ). All pixel measurements were translated to length metrics by means of the calibrated grid. We then cryogenically stored the tissue at −80°C in a 9:1 ratio of DMEM (VWR L0101-0500, Radnor, PA, USA):DMSO (VWR BDH1115-1LP) with protease inhibitor (ThermoFisher, A32953, Weltham, MA, USA) until further testing. Once ready to be tested, we rapidly thawed the vials in room temperature water and removed all chordae tendineae from the ventricularis surface of the anterior leaflets prior to tissue sample collection for testing.
10
Cell Lysis and Immunoprecipitation Protocol
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Cells were rinsed with ice-cold phosphate-buffered saline (PBS) and lysed with EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) or Triton buffer (40 mM HEPES pH 7.4, 150 mM NaCl, 2.5 mM MgCl2, and 1% Triton) supplemented with protease inhibitor (Thermo Fisher, A32953) and phosphatase inhibitors (phosphatase inhibitor cocktail sets I and II, Calbiochem). The cell lysates were centrifuged at 13,200 r.p.m. for 10 min at 4 °C. The protein concentrations were measured using Nanodrop (Thermo Fisher) with Bio-Rad protein assay reagent. Equal amounts of whole cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with the indicated antibodies. For immunoprecipitation, the lysates (1000–3000 μg) were incubated with 50% slurry of agarose conjugated antibody for 3–5 h at 4 °C. The immunoprecipitates were washed four times with NETN buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.5% NP-40) or Triton buffer before being resolved by SDS-PAGE. Anti-FLAG agarose beads (A2220) and anti-HA agarose beads (A2095) were purchased from Sigma. Anti-AKT1 antibody conjugated to agarose (sc-5298 AC) and anti-PRMT5 antibody conjugated to agarose (sc-376937 AC) were purchased from Santa Cruz Biotechnology.
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