Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, USA). Ursolic acid (UA) was purchased from Nanjing Zelang Plant Extract Co., Ltd. (Nanjing, China). Epirubicin was purchased from Rhawn (Shanghai, China). Autophagy inhibitor, 3-Methyladenine (3-MA), was purchased from Selleck Chemicals (Houston, TX, USA). Protease inhibitor, ECL luminescence reagent, Cell lysis buffer, Cell Counting Kit-8, Caspase3 ELISA Kit, Bradford Protein Assay Kit and autophagy agonist, rapamycin (RAPA), were obtained from Solarbio (Beijing, China). Class I PI3K, AKT, Beclin-1, LC3-II/LC3-, Atg5, Atg7 and β-Actin antibodies were purchased from CST (USA). RIPA Lysis Buffer (RIPA) was purchased from Biosharp (Guangzhou, China). Monodansylcadaverine (MDC) and Transwell chambers were purchased from Sigma (Shanghai, China). BCA Protein Assay Kit (BCA) was purchased from Bioteke (Beijing, China).
Cell lysis buffer
Manufactured by Solarbio
Sourced in China
Cell lysis buffer is a solution used to break open cells and release their contents, including proteins, nucleic acids, and other cellular components. It is a fundamental tool in molecular biology and biochemistry research.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Complete protease inhibitor mixture, by Solarbio (3 mentions)
Caspase3 elisa kit, by Solarbio (2 mentions)
Blocking solution, by Beyotime (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ipvh00010, by Merck Group (2 mentions)
Bca protein assay kit, by Applygen (2 mentions)
Bca kit, by Solarbio (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Monodansylcadaverine mdc, by Merck Group (1 mentions)
Transwell chamber, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ecl luminescence reagent, by Solarbio (1 mentions)
Rapamycin rapa, by Solarbio (1 mentions)
3 methyladenine 3 ma, by Selleck Chemicals (1 mentions)
Cell counting kit 8 (cck8), by Solarbio (1 mentions)
Bradford protein assay kit, by Solarbio (1 mentions)
Bca protein assay kit, by BioTeke (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Enhanced chemiluminescence, by PerkinElmer (1 mentions)
36 protocols using cell lysis buffer
1
Ursolic Acid Inhibits Autophagy in Cells
2
Quantifying CD44 Expression in Glioma Tissues
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The glioma tissues were homogenized and lysed with cell lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing 1% PMSF. The lysates were centrifuged at 12,000 × g for 5 min at 4°C. The protein concentration was determined using a bicinchoninic acid assay. The proteins (30 µg total protein/lane) were separated using SDS-PAGE on a 12% gel. Proteins were transferred to PVDF membranes (EMD Millipore), blocked with 5% fat-free milk for 25°C for 1 h and incubated with primary antibodies against CD44 (cat. no. D190741; 1:750; Sangon Biotech Co., Ltd.) and GAPDH (cat. no. TA-08; 1:1,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) at 4°C overnight. The membranes were washed with PBS and 0.1% Tween, and incubated with goat anti-mouse secondary antibody (cat. no. abs20001; 1:2,000; Absin Bioscience Inc.) at 25°C for 1 h. Protein bands were visualized using the enhanced chemiluminescence (PerkinElmer, Inc.) method and a Bio-Rad gel imaging system (Bio-Rad Laboratories, Inc.), and densitometry analysis was performed using ImageJ version 1.8.0 software (National Institutes of Health).
3
Western Blot Analysis of Cell Signaling Proteins
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Total protein was extracted from CRC cells using cell lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), and the BCA method was used to detect the protein. Total protein (30 µg/lane) was separated by 12% SDS-PAGE and the separated proteins were subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore). The PVDF membranes were blocked in blocking solution (Beyotime Institute of Biotechnology) for 60 min in a shaker at room temperature and incubated with primary antibodies against: Cyclin-dependent kinase 4 (CDK4; cat. no. ab137675), cyclin D (cat. no. ab62151), cyclin E (cat. no. ab33911), matrix metalloproteinase 9 (MMP9; cat. no. BA0573) and β-actin (cat. no. 4ab010745) overnight at 4°C on a shaker, all of which used primary antibody dilution buffer and were purchased from Beyotime Institute of Biotechnology. Following the primary incubation, membranes were incubated with HRP-conjugated affinipure rabbit anti-Goat IgG (H+L) (1:5,000; cat. no. SA00001-1; ProteinTech Group, Inc) at room temperature for 60 min. Protein bands were visualized using the bright ECL kit (Advansta, Inc. K-12045-D20, http://advansta.com/products/western-blot-substrate-WesternBright-ECL ) and protein bands were detected using an Odyssey Infrared Imaging system (LI-COR Biosciences).
4
Immunoprecipitation of Hsp90, Akt1, p53, and Ubiquitin
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Cells were homogenized in cell lysis buffer (Solarbio), supplemented with 1 mM PMSF and complete protease inhibitor mixture (Solarbio). The homogenate was centrifuged at 11,000 rpm/min for 15 min at 4°C. The supernatant was incubated with mouse-anti-human Hsp90 (Proteintech), rabbit-anti-human Akt1 (CST), p53, or ubiquitin (Proteintech) crosslinked beads at 4°C overnight with rotation. The pretreatment of beads and the immunoprecipitation step were carried out according to the PierceTM Crosslink Magnetic IP Kit instruction. The identification of the associated proteins was detected by western blot. Homogenates from co-culture cells were used as positive controls.
5
Quantitative Analysis of Oxidative Stress
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The cartilage tissues and chondrocytes were lysed using Cell lysis buffer (Solarbio, China) and detected with BCA kit (Servicebio, China). The MDA and GSH concentration were detected using MDA Assay Kit (Beyotime, China) or GSH/GSSG Assay Kit (Beyotime, China) and normalized based on the protein concentration. The Fe2+ detection was performed using Iron Assay Kit (Dojindo, Japan) according to the kit’s instructions.
6
Western Blot Analysis of Liver Tissue
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Powdered samples of HCC tumor or normal liver tissues or cells were lysed in cell lysis buffer (Solarbio, J619) containing a protease inhibitor cocktail (Roche, 4693124001). Total protein levels were quantified by bicinchoninic acid (BCA) assay Kit (Pierce, 23228). Twenty to forty microgram of total protein were resolved by SDS-polyacrylamide gels, transferred to nitrocellulose membranes (Millipore, HATF00010), and detected with the appropriate primary and HRP-conjugated secondary antibody. Prestained protein ladders (Thermo, 26619) were loaded to one well of each SDS-PAGE gel on western blots. Antibody detection was performed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, 34080) and imaged on the Molecular Imager ChemiDox XRS System from Bio-Rad. Primary anti-AGRN (PA5-37121) was purchased from Thermofisher, anti-VWA1 (14322-1-AP), anti-CDH2 (22018-1-AP), anti-VIM (10366-1-AP) and anti-GFP (50430-2-AP) were purchased from Proteintech, anti-α-tubulin (ab7291) and anti-β-actin (ab8227) antibodies were purchased from abcam. Secondary anti-rabbit (#31466) and anti-mouse (#31431) antibodies were purchased from Pierce Biotechnology.
7
Microglia Response to LPS and ASD
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The dentate gyrus was dissociated from slices containing the hippocampus, flash‐frozen in liquid nitrogen, and homogenized. Primary microglia were cultured in six‐well plates at 5 × 105 cells/cm2 and then treated for 24 or 48 h with LPS or PBS in the presence or absence of ASD. The culture medium was collected, microglia were lysed in cell lysis buffer (Solarbio), and the lysates were centrifuged at 1000 g for 30 min. The concentration of total protein in the supernatant was determined using the BCA kit (BOSTER), and each sample was diluted to 1 g/mL. Then, samples were assayed using commercial ELISAs against the following signaling factors: interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, IL‐10, IL‐4, insulin‐like growth factor (IGF)‐1, and brain‐derived neurotrophic factor (BDNF; BOSTER); inducible nitric oxide synthase (iNOS) and arginase (Arg‐1; Elabscience); transforming growth factor (TGF)‐β (4A Biotech); and basic fibroblast growth factor (bFGF), epidermal growth factor (EGF; ColorfulGene Biotech), and nerve growth factor (NGF; BOSTER). The manufacturer‐specified detection limit of all kits was 1–4 pg/mL.
8
Protein Extraction and Western Blot Analysis
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The total protein was extracted with cell lysis buffer (Solarbio cat#R0010) followed by the addition of protease and phosphorylated protease inhibitors. The protein concentration was determined using a spectrophotometer (Thermo Fisher). Next, 10 μL of total protein was loaded onto 6%–12% SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated with primary and secondary antibodies. Protein signals were detected using the ECL method. Primary antibodies used in this study included anti-p-AMPK (1 : 1000, Abcam32047), anti- AMPK (1 : 1000, Abcam133448), anti-p62 (1 : 1000, Ab109012), anti-beclin-1 (1 : 1000, Ab210498), anti-mTOR (1 : 1000, Abcam134903), anti-p-mTOR (1 : 1000, Abcam109268), anti-caspase-3 (1 : 1000, Ab13847), anti-caspase-9 (1 : 1000, Ab184786), and anti-LC3-II (1 : 1000, Ab192890). All primary antibodies were incubated overnight at 4°C. The HRP-labeled goat anti-rabbit lgG H&L (1 : 10,000) was incubated for 2 h at room temperature.
9
Protein Extraction and Western Blot Analysis
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Total protein was extracted with cell lysis buffer (Solarbio, Beijing, China). Proteins were separated by 10% SDS‐PAGE and then transferred to nitrocellulose membranes (Millipore). The membranes were blocked with 5% non‐fat milk (Sangon Biotech, Shanghai, China) for 2 h, then incubated with primary antibodies overnight at 4°C, and finally incubated with HRP‐conjugated secondary antibodies for 2 h at room temperature. Next, immunoreactive bands were detected using an enhanced chemiluminescence kit (Bio‐Rad, Hercules, CA, USA). β‐Actin was used as a loading control. The primary antibodies included mouse anti‐MAP2 (Millipore; 1:1000), mouse anti‐Tuj1 (Millipore; 1:1000), rabbit anti‐GFAP (Millipore, 1:1000), rabbit anti‐Acsl4 (Abcam; 1:1000), rabbit anti‐Akt (Cell Signaling Technology, Danvers, MA, USA; 1:1000), rabbit anti‐phospho‐Akt (Cell Signaling Technology; 1:1,000), rabbit anti‐PI3K (Abcam; 1:1000) and mouse anti‐β‐actin (Abcam; 1:1000).
10
Immunoprecipitation of ASC/HAX-1/NLRP3 Complexes
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Cells were homogenized in cell lysis buffer (Solarbio) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and a complete protease inhibitor mixture (Solarbio). The homogenate was then centrifuged at 11,000 rpm for 15 min at 4 °C. Next, the supernatant was incubated with ASC/HAX-1/NLRP3 antibody or rabbit/mouse normal IgG crosslinked beads at 4 °C for 1 h with rotation. The pretreatment of beads and the immunoprecipitation steps were carried out with a Pierce Crosslink Magnetic IP Kit in accordance with the manufacturer’s instructions. Proteins were identified by western blotting or protein mass spectrometry. Homogenates from tissue or cells were used as positive controls. Three individual replicates in every group were contained in Co-IP assays.
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