Cells were washed with ice-cold PBS and centrifuged at 400 × g for 5 min at 4 °C. The resulting pellet was lysed with RIPA buffer (KeyGEN) supplemented with 1x protease/phosphatase inhibitor (Solarbio). The homogenate was cleared by centrifugation at 4 °C for 10 min at 16,000 × g, and the supernatant containing the protein fraction recovered. Samples were subjected to electrophoresis in SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were blocked with 1% non-fat milk and incubated with primary antibodies overnight, followed by 1-hour incubation with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies at room temperature and washed 3 times. The following primary antibodies were used: anti-p-p38 MAPK (Cat.#YP0338), anti-p38 (Cat.#YT3513) (Immunoway), anti-AKT (S473) (Cat.#A5030), anti-AKT (Cat.# 10176-2-AP) (Cell Signalling Technology), anti-CDK1 (Cat.# CY516-50), anti-GAPDH (Cat.# 12231 P) (Abways), and anti-cyclin B1(Santa Cruz Biotechnology). Antigen-specific binding of antibodies was detected with the ECL (enhanced chemiluminescence) Plus reagents (Beyotime). Protein bands were quantified ImageJ software (Wright Cell Imaging Facility) and then normalized to the GAPDH loading control. For full scans of Western blots see Supplemental Figures S1–S2 .
Protease phosphatase inhibitor
Manufactured by Solarbio
Sourced in China
Protease phosphatase inhibitor is a lab equipment product used to inhibit the activity of proteases and phosphatases in biological samples. It is designed to preserve the integrity of proteins and protein modifications during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Anti akts473, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti cdk1, by Cell Signaling Technology (2 mentions)
Anti p p38 mapk, by Immunoway (2 mentions)
Anti p38, by Immunoway (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti cyclin b1, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Keygen Biotech (2 mentions)
Plus reagents, by Beyotime (2 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
Ripa buffer high, by Solarbio (1 mentions)
Bca protein quantification kit, by Vazyme (1 mentions)
Nucleoprotein extraction kit, by Solarbio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Solarbio (1 mentions)
Penicillin streptomycin mixture, by Solarbio (1 mentions)
Bca protein quantitative kit, by Solarbio (1 mentions)
Sorvall legend micro 21r microcentrifuge, by Thermo Fisher Scientific (1 mentions)
6 protocols using protease phosphatase inhibitor
1
Quantitative Western Blot Analysis
2
Western Blot Analysis of Signaling Proteins
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Cells were washed with ice-cold PBS and centrifuged at 400 × g for 5 min at 4 °C. The resulting pellet was lysed with RIPA buffer (KeyGEN) supplemented with 1x protease/phosphatase inhibitor (Solarbio). The homogenate was cleared by centrifugation at 4 °C for 10 min at 16,000 × g, and the supernatant containing the protein fraction recovered. Samples were subjected to electrophoresis in SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were blocked with 1% non-fat milk and incubated with primary antibodies overnight, followed by 1-hour incubation with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies at room temperature and washed 3 times. The following primary antibodies were used: anti-p-p38 MAPK (Cat.#YP0338), anti-p38 (Cat.#YT3513) (Immunoway), anti-AKT (S473) (Cat.#A5030), anti-AKT (Cat.# 10176-2-AP) (Cell Signalling Technology), anti-CDK1 (Cat.# CY516-50), anti-GAPDH (Cat.# 12231 P) (Abways), and anti-cyclin B1(Santa Cruz Biotechnology). Antigen-specific binding of antibodies was detected with the ECL (enhanced chemiluminescence) Plus reagents (Beyotime). Protein bands were quantified ImageJ software (Wright Cell Imaging Facility) and then normalized to the GAPDH loading control. For full scans of Western blots see Supplemental Figures S1–S2 .
3
Spinal Cord Protein Extraction Protocol
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Animals were deeply anesthetized with 1% pentobarbital sodium (0.1 g/kg, i.p.) and the ipsilateral spinal cord in the lumbar enlargement site was rapidly separated and homogenized in ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride and protease phosphatase inhibitor (Solarbio Company, China). After being kept on ice for 1 h, the lysates were centrifuged at 12,000 rpm for 15 min at 4 °C and the supernatants mixing with loading buffer were denatured at 95 °C for 5 min. The protein concentrations were measured using a bicinchoninic acid (BCA) kit (Pierce Biotechnology Inc.).
4
Western Blot Analysis of Skeletal Muscle Proteins
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The skeletal muscle was lysed in RIPA buffer (High) (Solarbio, Wuhan, China) containing one mM phenylmethylsulfonyl fluoride (PFMS) and 1× protease phosphatase inhibitor (Solarbio, Wuhan, China). After quantification with a BCA protein quantification kit (Vazyme, Nanjing, China), 20 g of protein lysate was loaded onto an SDS‒PAGE gel. The PAGE gel was cut according to the molecular weight of the target protein, and the protein was transferred to a PVDF membrane after electrophoresis. The membranes were blocked with 5% nonfat milk at room temperature and incubated with primary antibody at 4 °C overnight. See Table S2 for specific information on the primary antibodies. After washing in PBST, the membranes were incubated with a goat anti-rabbit IgG-HRP secondary antibody (1:10,000, Absin, Shanghai, China). Proteins were detected using a gel imaging system (Tanon, Shanghai, China). Immunoreactive bands were visualized and quantified using ImageJ software.
5
Anti-inflammatory Mechanisms of LPS-induced RAW264.7 Cells
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The following reagents were used in this research: RAW264.7 cells were purchased from the Typical Culture Preservation Committee Cell Bank, Chinese Academy of Sciences. DMEM (Solarbio, Beijing, China). FBS (Gibco, GrandIsland, USA). Penicillin-streptomycin mixture, Nucleoprotein extraction kit, BCA protein quantitative kit and protease phosphatase inhibitor (SolarBio, Beijing, China). LPS (Sigma-Aldrich, St. Louis, MO, USA). NO kit and ROS kit (Beyotime Biotechnology Shanghai, China). Mouse TNF-α Enzyme-linked immunosorbent assay (ELISA) kit and mouse IL-6 ELISA kit (4A Biotech Co, Ltd. Beijing, China). Evo M-MLV RT Kit with gDNA Clean and SYBG Green Premix Pro Taq HS qPCR kit (Tli RNadeH Plus, Accurate Biotechnology, Hunan, China). Antibody iNOS, COX-2, NF-κB P65, p-NF-κB P65 (Cell Signaling, USA). Primers were designed and synthesized by Thermo Fisher Scientific (Shanghai, China). The primers were shown in Supplementary Table 1 .
6
Luteolin and Oxaliplatin Cytotoxicity Assay
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A total of 3×106 MFC cells were inoculated into 100-mm culture dishes. After overnight culture, the cells were incubated with luteolin (20 µM) and/or oxaliplatin (5 µM) (total volume 7 ml/well) for 24 h. Then, the cells were rinsed with PBS and lysed on ice for 30 min with RIPA buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) containing 0.1 M PMSF (cat. no. P0100; Beijing Solarbio Science & Technology Co., Ltd.) and protease phosphatase inhibitor (cat. no. P1261; Beijing Solarbio Science & Technology Co., Ltd.). The cell lysate was centrifuged at 12,000 × g (Thermo Scientific™ Sorvall™ Legend™ Micro 21R Microcentrifuge; Thermo Fisher Scientific, Inc.) for 15 min at 4°C, and the supernatant was collected for the subsequent tests. The protein quantification was performed using the BCA protein assay kit (cat. no. PC0020; Beijing Solarbio Science & Technology Co., Ltd.) (30 (link)). The data were obtained at the absorbance of 562 nm by a microplate reader (Infinite 200 PRO, Tecan, Tecan Austria GmbH). The amount of protein was calculated in accordance with the prescribed computational formula of the kit's protocol.
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