Cells or exosomes were homogenized by the RIPA lysis buffer containing a cocktail of protease inhibitors (Thermo). The total protein (30 μg) was resolved in SDS-PAGE gels and shifted to the NC membranes, followed by incubation with primary antibodies and subsequent corresponding secondary antibodies. The bands were visualized by incubating with an enhanced chemiluminescence (ECL) reagent (Millipore, USA). ELISA kits were ordered from Thermo to detect the secretion of IL-1β and IL-18 in BALF under the manufacturer's instructions.
Cocktail of protease inhibitor
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The Cocktail of protease inhibitors is a laboratory product designed to inhibit the activity of proteases, which are enzymes that break down proteins. This product is commonly used in research applications to prevent protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Merck Group (3 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Vibra cell, by Sonics (2 mentions)
Ecl reagent, by Merck Group (1 mentions)
Gel image system, by Bio-Rad (1 mentions)
Ecl solution, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Nitrocellulose membranes, by Absin (1 mentions)
Sw55ti rotor, by Beckman Coulter (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Rabbit anti β actin antibody, by Cell Signaling Technology (1 mentions)
Af808, by R&D Systems (1 mentions)
Nonidet p 40, by Roche (1 mentions)
Mouse monoclonal anti cd63, by Abcam (1 mentions)
Mouse monoclonal anti alix, by Abcam (1 mentions)
Mouse monoclonal anti tsg101, by Abcam (1 mentions)
16 protocols using cocktail of protease inhibitor
1
Western Blot and ELISA Analysis
2
Quantifying GPX4 in Liver Cancer Cells
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Liver cancer cells were lysed by ice-cold RIPA buffer (Thermo, USA) and added with a cocktail of protease inhibitors (Thermo). The lysates were then subjected to the SDS-PAGE and shifted to NC membranes. The protein bands were interacted with primary antibodies against GPX4 (1:1000, Abcam, USA) and β-actin (1:1000, Abcam), followed by secondary anti-mouse and anti-rabbit antibodies (1:2000, Abcam). The bands were then visualized by an ECL solution (Millipore, Germany) in a Gel Image system (Bio-Rad).
3
Western Blot Protein Detection
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Cells with the indicated treatment were lysed by ice-cold RIPA buffer (Thermo, USA) added with a cocktail of protease inhibitors (Thermo). The lysates were then subjected to the SDS-PAGE and shifted to nitrocellulose membranes (absin, China). The protein bands were interacted with primary antibodies at room temperature for 2 hours and 4°C overnight, followed by secondary anti-mouse and anti-rabbit antibodies. The bands were then visualized by using an Odyssey in an Odyssey software (LI-COR, USA).
4
Lipid Raft Isolation from Porcine Alveolar Macrophages
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Low-density detergent-insoluble lipid raft fractions were isolated as described previously with modifications (Chung et al., 2005 (link)). Briefly, PAMs were scraped with a rubber policeman into ice-cold PBS and collected by centrifugation at 1000g for 3 min at 4 °C. Then pelleted cells were lysed using 0.3 ml of ice-cold TNE buffer (25 mM Tris [pH 7.5], 150 mM NaCl, 5 mM EDTA) containing 1% Triton X-100 (Merck), 1 mM NaF, and a cocktail of protease inhibitors (Thermo) at 4 °C for 30 min with gentle agitation. Then, the cell lysates were centrifuged at 4 °C for 10 min at 3500g to remove nuclei and insoluble materials. The supernatants were collected and mixed with equal volumes of 80% (wt/vol) sucrose in TNE buffer and then placed in the bottoms of ultracentrifuge tubes. A discontinuous sucrose gradient was formed by sequentially overlaying 2.1 ml of 30% and 1.2 ml of 5% sucrose in TNE buffer. These mixtures were centrifuged at 4 °C for 18 h at 250,000g in a SW 55 Ti rotor (Beckman). After centrifugation, lipid rafts could be visible as an opaque band at the boundary between the 5% and 30% sucrose solutions. A total of 11 gradient fractions were collected from the top to bottom and stored at −80 °C until use.
5
Immunoblotting for SPP1 in Melanocytes
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Single sorted melanocytes or cells from mouse whole-back skin were lysed in RIPA buffer (Sigma) containing a cocktail of protease inhibitors (Thermo Fisher). Of each cell lysate, 25 µg was loaded onto a 12% separating Bis-Tris gel. Proteins were transferred to a nitrocellulose membrane. Membrane was incubated with primary goat anti-mouse SPP1 antibody (1:100; AF808, R&D) or rabbit anti-β-actin antibody (1:1,000; 4967, Cell Signaling) at a concentration of 2.5 μg ml−1. The blot was developed with Enhanced Chemiluminescence Plus Developer (Fisher Scientific).
6
Western Blot Protein Analysis
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Western blot analysis was carried out by preparing total protein lysates with RIPA lysis buffer (Thermo Fisher) and a cocktail of protease inhibitors (Thermo Fisher). 8% SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to resolve and separate 30 μg of total cellular proteins. Proteins are transferred to a nitrocellulose membrane. Membranes were blocked in 5% skimmed milk. The membranes were then incubated with the appropriate primary and secondary antibodies overnight at 4 °C.
7
Biological Sample Preparation for Biochemical Analysis
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After the end of behavioral studies, the animals were instantly decapitated, and the following biological material was obtained: post-decapitation blood; and the ipsi- and contralateral regions of the brain—the hippocampus (whole structure from each hemisphere) and frontal cortex (FC, +6.1–3.0 mm from bregma). Subsequently, the blood was centrifuged 1500× g at 40 °C for 15 min to obtain serum. The isolated brain regions were homogenized in a Potter homogenizer using a 10-fold excess of a cold standard phosphate–salt buffer containing 0.1% nonidet P-40(Roche) and a cocktail of protease inhibitors (Thermo Scientific, Waltham, MA, USA) with 10 impacts of the pestle at a rotation speed of 1000 rpm. The homogenates were centrifuged at 13,000× g at 40 °C for 30 min to obtain a soluble protein fraction (supernatant), which was aliquoted and stored at −80 °C before biochemical studies. The samples were recoded for the following biochemical assay.
8
Extracellular Vesicle Protein Profiling
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PEX preparations were lysed in RIPA buffer (Sigma-Aldrich, USA) containing a cocktail of protease inhibitors (Thermo, USA). Proteins were denatured in 2 × SDS buffer at 95°C, separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, USA). After blocking with 5% milk powder for 1 h at room temperature, the membranes were incubated with the antibodies as follows: mouse monoclonal anti-TSG101 (1:500, Abcam, UK), mouse monoclonal anti-CD63 (1:1000, Abcam, UK), mouse monoclonal anti-ALIX (1:1000, Abcam, UK), and mouse monoclonal anti-GAPDH (1:2000, Abcam, UK) as the control. The samples were incubated with a secondary goat anti-mouse antibody (1:5000, Pierce, USA) for 1 h at room temperature. The blots were visualized using a GE Amersham Imager 600.
9
Western Blot Analysis of Dental Proteins
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Cells were lysed in RIPA buffer (Beyotime, China) combined with a cocktail of protease inhibitors (Thermo Scientific, Rockford, IL). Equal amounts of proteins (25 μg) of different groups were separated by 10% SDS-PAGE and transferred to 0.45-μm PVDF membranes (Millipore, USA). The membrane was first blocked with 5% BSA for 1 h at room temperature and incubated at 4 °C overnight with primary antibodies: GAPDH (1:1000; Abcam, Cambridge, UK), RUNX2(1:1000; Cell Signaling Technology, Danvers, MA), DSPP (1:1000; Abcam, Cambridge, UK), and DMP-1 (1:1000; Abcam, Cambridge, UK). After washed with Tris-buffer saline containing 0.05% Tween 20 (TBST) for three times and 5 min each, the membranes were incubated with secondary antibodies labeled with horseradish peroxidase (1:3000; Cell Signaling Technology, Danvers, MA) at room temperature for 1 h. Blots were visualized using ECL Western Blotting Substrate.
10
Western Blotting of STAT1 and p-STAT1
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Cell protein was extracted on ice using RIPA lysis buffer (Beyotime) in the presence of a freshly added cocktail of protease inhibitors (Thermo), and then quantified by the BCA method (Pierce). A total of 30 μg total protein per lane was loaded onto 12% SDS-polyacrylamide gels, electrophoresed, and then transferred to 0.2-μm nitrocellulose membranes (Pierce) for 1 h at 350 mA using a wet western blotting system. After blocking with 5% non-fat milk in PBS, the membrane was incubated with rabbit anti-STAT1 antibody (Epitomics, Cat. 2251–1) or rabbit anti-phospho-STAT1 (CST, Cat. 7649) overnight at 4 °C. IRDye 800CW goat anti-rabbit IgG (LI-COR, Cat. 926–32,211) was used as secondary antibody to display the signal, and rabbit anti-GAPDH (Epitomics, Cat. 2251–1) antibody was used as an internal standard.
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