SARS-CoV-2 RBD dodecamer mRNA was transfected into HEK293T cells by Lipofectamine™ 2000 according to the manufacturer's instructions (Thermo Fisher Scientific, 11668027, Waltham, MA, USA). Supernatants and cell lysates were obtained, and the expression of SARS-CoV-2 RBD dodecamer antigen in vivo was detected by SARS-CoV-2 RBD protein detection ELISA kit (Vazyme, 7E510D1, Nanjing, China) and Western blotting (WB). Briefly, cells were lysed with Cell Lysis Reagent (Sigma–Aldrich, C3228-50 ML, St. Louis, MO, USA) containing protease inhibitor (Thermo Fisher Scientific, 78,430, Waltham, MA, USA), denatured at 100 °C for 10 min, separated by electrophoresis on 10% SDS-PAGE gels, and then transferred to nitrocellulose transfer membranes (GE Amersham Biosciences, 10-6000-01, Pittsburgh, PA). Membranes were incubated with RBD primary Abs overnight (Proteintech Europe, 67758-1-Ig, Deansgate, UK), and then incubated with corresponding secondary Abs conjugated to HRP (Santa Cruz Biotechnology, sc-2005, Dallas, TX, USA). Finally, the relative expression levels of protein were detected using ECL reagents (Santa Cruz Biotechnology, sc-2048, Dallas, TX, USA) and quantified by Quantity One software [Bio-Rad Laboratories, Quantity One®1-D, Hercules, CA, USA)].
Cell lysis reagent
Manufactured by Merck Group
Sourced in United States
The Cell Lysis Reagent is a laboratory product designed to facilitate the disruption and lysis of cells. It is a solution formulated to break down the cellular membrane and release the contents of the cell, including proteins, nucleic acids, and other intracellular components. This reagent is commonly used in various cell biology and molecular biology applications that require the extraction and isolation of cellular components for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (5 mentions)
Anti vimentin, by Santa Cruz Biotechnology (3 mentions)
Anti e cadherin, by Santa Cruz Biotechnology (3 mentions)
Anti n cadherin, by Santa Cruz Biotechnology (3 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Luciferase reagent, by Molecular Devices (2 mentions)
Gemini ex, by Molecular Devices (2 mentions)
Atp bioluminescence assay kit, by Roche (2 mentions)
Anti gapdh, by Abcam (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Ecl reagent, by Santa Cruz Biotechnology (1 mentions)
Quantity one 1 d, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Immobilon membrane, by Merck Group (1 mentions)
40 protocols using cell lysis reagent
1
SARS-CoV-2 RBD Dodecamer Antigen Expression in HEK293T Cells
2
Immunofluorescence Analysis of S100A14 and S100A16
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Cultured cells were plated on 8-well chamber slides for 24 to 48 h. Cells were fixed with 4% paraformaldehyde and permeabilized for 5 min at room temperature with 0.1% Triton X-100 in PBS, followed by blocking for 1 h with 5% skimmed milk. Cells were stained with rabbit polyclonal antibodies for S100A14 and S100A16 for overnight at 4°C, followed by incubation with FITC-labeled secondary antibody for 1 h at room temperature. To observe filamentous actin, cells were stained with rhodamine-conjugated phalloidin (Molecular Probes). The slides were mounted in mounting medium containing DAPI and analyzed using an inverted fluorescence microscope and by laser scanning confocal microscopy. Cellular proteins extracted with Cell lysis reagent (Sigma) were analyzed by Western blotting. Equal amounts of proteins were electrophoresed by standard SDS-PAGE under reducing conditions and were then transferred onto Immobilon membranes (Merck-Millipore). The target proteins were detected by immunoblotting using S100A14 and S100A16 antibodies (diluted 1/1,000) according to standard protocols using the ECL Advance Western Blotting Detection Kit (GE Healthcare).
3
Protein Expression Analysis via Western Blot
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Whole cell extracts were prepared with a cell lysis reagent (Sigma-Aldrich, St Louis, MO, USA) according to the manual, and then the protein was quantified by a bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). The protein samples were later separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10 %) and detected by Western blot using the following specific antibodies: anti-HER2, COX-2, P-ERK, ERK, AKT, p-AKT, MMP-2, caspase-3, and β-actin polyclonal antibodies (Santa Cruz Biotechnology Inc., Dallas, TX, USA), the antibodies were suspended in 5% BSA. Goat anti-rabbit IgG (Pierce) secondary antibody conjugated to horseradish peroxidase and ECL detection systems (SuperSignal West Femto, Pierce) were used for detection.
4
Protein Extraction and Western Blot Analysis
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After treatment, cells were washed twice with cold PBS and then pelleted, and then cell lysis reagent (Sigma-Aldrich) was added to the cell pellet for protein extraction. Protein concentration was determined by bovine serum albumin (BSA) assay using BSA as the reference, and the absorbance of the reacted product was measured at wavelength of 540–595 nm. Samples were boiled for 3 min before loading onto 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel. After electrophoresis, the gels were electro blotted onto a polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Whitehouse Station, NJ, USA). The antibodies used were as follows: anti-Bcl-2 rabbit polyclonal antibody (Abcam, Cambridge, MA, USA), anti-Bax rabbit polyclonal antibody (Abcam), anti-β-actin rabbit polyclonal antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA), and anti-rabbit IgG HRP (Abcam). The blot was visualized with an enhanced chemiluminescence (ECL) kit (Merck Millipore) and exposed to ECL Hyperfilm.
5
Insulin and Glucose Regulation Proteins
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The expressions of insulin, GLUT2, PGC-1α, GCK, and G6Pase were determined by western blot and GAPDH was employed as control. Pancreas was lysed using cell lysis reagent (Sigma) and phosphatase inhibitors (Sigma) and lysed by Tissue Homogenizer (Bertin Technologies, France). The crude lysate was transferred to new Eppendorf tubes. Each sample was added to 20 μL 2x sample loading buffer (0.125 M of 5 M Tris-HCl, Amresco; 20% glycerol, Usb; 4% of 10% sodium dodecyl sulfate, Amresco; 1% β-mercaptoethanol, Amresco; 0.2% of 0.05% (w/v) bromophenol blue, Sigma) and boiled for 5 min before loading. Proteins were separated by SDS-PAGE, transferred to immobilon P membrane (Millipore), and probed with different antibodies as indicated. All antibodies were purchased from Cell Signaling (Beverly, MA). The results were visualized using ECL kit (Abcam) and observed by GeneGnome machine (Syngene).
6
Western Blotting of Bladder Tissues
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Western blotting was performed as described previously.26 Protein lysates were obtained by homogenizing normal bladder tissues, organoid samples, and 2D urothelial carcinoma cell lines with Cell Lysis Reagent (Sigma‐Aldrich) containing 1% protease inhibitor cocktail (Sigma‐Aldrich). Loading proteins (10 μg) were separated by SDS‐PAGE (7.5%) and transferred to a nitrocellulose membrane (Wako). After blocking with 0.5% skimmed milk, the membranes were incubated with primary antibody (CK5; 1:500, VCP; 1:500) at 4°C overnight followed by incubation with the secondary antibody (1:10 000 dilution, 1 h) and ECL Prime (GE Healthcare). Results were obtained with FujiFilm LAS‐3000 and quantified using ImageJ densitometry analysis software (National Institutes of Health).
7
ATP Content Measurement Protocol
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The measurement of ATP content was analyzed using ATP bioluminescence assay kit (11699709001; Roche Life Science, Indianapolis, IN, USA) according to the manufacturer's instructions. After 2‐h reperfusion, the PRNCs were incubated with cell lysis reagent (Sigma, Fremont, CA, USA) and protease inhibitor cocktail (Sigma) to the samples for 5 min at room temperature in darkness. Luciferase reagent was added to the samples, and the luminescence of the live cells was measured by the Gemini EX florescence plate reader (Molecular Device).
8
Quantitative Analysis of IL-4 and IL-9
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Protein extracts were prepared with a cell lysis reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manual, and the protein was quantified by a BCA assay (Pierce, Rockford, IL, USA). Then, the protein samples were separated by SDS-PAGE (10%) and detected by Western blot using polyclonal (rabbit) anti-IL-4, anti-IL-9 and anti-GAPDH (Abcam, USA) antibodies. Goat anti-rabbit IgG (Pierce, Rockford,IL, USA) secondary antibody conjugated to horseradish peroxidase and ECL detection systems (SuperSignal West Femto, Pierce) were used for detection. IL-4 and IL-9 levels were analyzed using ELISA kits for IL-4 (eBioscience, USA) and IL-9 (GUSABIO, USA) according to the manufacturer’s protocols.
9
ATP Content Measurement in PRNCs
10
Quantitative Western Blot Analysis
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Western blot analysis was done according to published methods [28 (link),29 (link)]. Briefly, the lung tissues or cells were extracted with cell lysis reagent (Sigma, St. Louis, MO, USA ). Then, protein samples were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The PVDF membrane was incubated in 5% bovine serum albumin (BSA) for 2 h. After 3 washes with Tris-buffered saline containing Tween-20 (TBST), the PVDF membrane was incubated with E-cadherin, β-actin, P-ERK1/2 and ERK1/2 antibodies (Cell Signaling Technology, Danvers, USA) separately overnight at 4°C. The PVDF membrane was then washed with TBST 3 times followed by incubation with HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, USA) for 2 h at room temperature. After 3 washes with TBST, immunoreactive bands were visualized using chemiluminescence detection reagents (Bio-Rad Laboratories, Redmond, USA). The signal intensities of bands were quantified by using ImageJ software.
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