Total protein was extracted using RIPA lysis buffer containing protease inhibitors (APExBIO Technology, Houston, USA). Protein concentrations were determined using BCA Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Then 10% SDS-PAGE gel electrophoresis was carried out using 50 μg of protein from each sample. After gel transfer, PVDF membranes were incubated with primary antibodies including rabbit anti-Bax (1:1,000, Proteintech, Wuhan, China), Bcl-2 (1:1,000, Proteintech, Wuhan, China), survivin (1:1,000, Proteintech, Wuhan, China), cleaved caspase-3 (1:1,000, Proteintech, Wuhan, China), cleaved caspase-9 (1:1,000, Proteintech, Wuhan, China), p21 (1:1,000, Proteintech, Wuhan, China), cyclin D1 (1:1,000, Proteintech, Wuhan, China), IRS1(1:1,000, Affinity, USA), p-PI3K (1:1,000, Affinity, USA), p-AKT (1:1,000, Affinity, USA), PI3K (1:1,000, Affinity, USA), and AKT (1:1,000, Affinity, USA) overnight at 4°C. After washing, membranes were further incubated with fluorochrome-labeled anti-rabbit IgG secondary antibody (1:10,000, Proteintech, Wuhan, China) for 1 h at room temperature. The membranes were imaged using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA), which was used to normalize relative expression level of each protein to GAPDH.
Protease inhibitor
Manufactured by Apexbio
Sourced in United States, China
Protease inhibitors are laboratory reagents used to prevent the activity of proteases, enzymes that catalyze the breakdown of proteins. They are commonly used in biological research to maintain the integrity of protein samples during extraction, purification, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor, by Apexbio (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Cleaved caspase 3, by Proteintech (1 mentions)
P akt, by Affinity Biosciences (1 mentions)
Cleaved caspase 9, by Proteintech (1 mentions)
Survivin, by Proteintech (1 mentions)
Rabbit anti bax, by Proteintech (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Cyclin d1, by Proteintech (1 mentions)
Bcl 2, by Proteintech (1 mentions)
P pi3k, by Affinity Biosciences (1 mentions)
Phospho smad2 3, by Cell Signaling Technology (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
β actin, by Affinity Biosciences (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Gapdh, by Affinity Biosciences (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Genegnome xrq imager, by Syngene (1 mentions)
11 protocols using protease inhibitor
1
Western Blot Analysis of Apoptosis and Proliferation Markers
2
Western Blot Analysis of Signaling Proteins
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Total protein was extracted from CD4+ T cells in the coculture system using RIPA lysis buffer (Solarbio, Beijing, China) containing protease inhibitors (APExBIO Technology LLC, USA). The protein samples were diluted to the same final total protein concentration and then subjected to 10% SDS‒PAGE; the proteins were transferred to polyvinylidene fluoride membranes (Merck Millipore, USA). Monoclonal antibodies for detecting SHP2 (1:1000) (Affinity, China), phospho-SHP2 (1:1000) (Affinity, China), SMAD2/3 (1:1000) (Cell Signaling Technology, USA), phospho-SMAD2/3 (1:1000) (Cell Signaling Technology, USA), GAPDH (1:1000) (Affinity, China) and β-actin (1:1000) (Affinity, China) were added to the membranes and incubated at 4 °C overnight. The membranes were washed and incubated with the corresponding diluted secondary horseradish peroxidase (HRP)-marked antibodies (1:2000) (Affinity, China) at room temperature for 1 h. Finally, the blots were developed with ECL solution and photographed with an exposure instrument.
3
Detecting IREB2-Ubiquitin Interactions
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All samples used for Co-IP were treated with Cell Lysis Buffer for western blotting and IP (Cat# P0013; Beyotime; China) supplemented with a protease inhibitor (Cat# K1019; APEXBIO, USA). Proteins containing IREB2 were precipitated using Protein A/G (Cat# L-1004; Biolinkedin, China) and the ubiquitin levels were detected by western blotting.
4
Protein Extraction and Western Blot Analysis
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Proteins were extracted from ground lung tissue or cultured cell samples by adding RIPA lysate (Solarbio Life Science, China). The protein concentration was determined with a PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) after an addition of a phosphatase inhibitor and a protease inhibitor (APExBIO Technology LLC, Houston, TX, USA) according to the volume of the sample. Proteins were heated to 100 °C for denaturation and then electrophoresed in 10% SDS-PAGE gels. The proteins were transferred with a 0.2 μm PVDF membrane (Millipore, Burlington, MA, USA) and blocked with 5% skimmed milk (Sigma-Aldrich, USA) for 2 h. After elution, the samples were incubated with the corresponding primary antibody at 4 °C overnight. After 1 h of secondary antibody (Proteintech, Shanghai, China) incubation at room temperature, the proteins were visualized with ECL luminescent solution (Cwbio, Taizhou, China) in a GeneGnome XRQ imager (Syngene, Cambridge, UK). The gray values of the bands were calculated and analyzed using ImageJ 1.5.2a software. The primary antibodies used in this experiment are shown in Table 1 .
5
Metabolite-Induced Protein Responses
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Cells were washed with cold phosphate buffered saline (PBS) for three times, scraped and centrifuged at 1,000 rpm for 5 min. The cell pellets were resuspended in mammalian protein extraction reagent (M-PER, Pierce/Thermo Fisher Scientific, cat. no. 78503) containing protease inhibitor (ApexBio Technology, Houston, TX, USA, cat. no. K1007) and phosphatase inhibitor (ApexBio Technology, cat. no. K1013), and lysed on ice for 30 min. The supernatants were collected by centrifugation at 18,000 g for 10 min. Protein concentration was determined by bicinchoninic acid (BCA) Protein Assay (Beyotime Biotechnology, Beijing, China, cat. no. P0011). The cell lysates were then diluted with M-PER lysis to 3 μg/μL and incubated with given metabolites or solvents for 1 hr at 25 ˚C. Specifically, the glycolytic metabolites were administered at dosages detailed as follows: D-Fructose 1,6-bisphosphate (FBP, 200 μM), D-fructose 6-phosphate (F6P, 100 μM), D-glucose 6phosphate (G6P, 100 μM), D-ribose 5-phosphate (R5P, 30 μM), D/L-glyceraldehyde-3-phosphate (G3P, 30 μM), D-2-phosphoglycerate (2PG, 10 μM) from Toronto Research Chemicals, North York, Ontario, Canada. D-3phosphoglycerate (3PG, 10 μM), phosphoenolpyruvate (PEP, 5 μM), pyruvate (Pyr, 300 μM), L-lactate (Lac, 2 mM). The dosages were set based on the intracellular concentrations of these metabolites as reported in literature 42, 43 .
6
Western Blot Analysis of EMT Markers
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U2OS and Soas2 cells were treated with RIPA buffer (Sigma‐Aldrich) with protease inhibitor (ApexBio Technology) for total protein extraction. Protein (30 μg) was separated by 12% SDS‐PAGE and transferred onto PVDF membranes. After blocking with 5% skimmed milk, the membranes were incubated overnight with primary antibodies against E‐cadherin (ab231303, 1 μg/mL; Abcam), N‐cadherin (ab76011, 1:5000), Slug (ab27568, 1:500), HK2 (ab209847, 1:10000), Twist (ab50887, 2 μg/mL), and GAPDH (ab9485, 1:2500) at 4°C followed by a 2‐h incubation with secondary antibodies at room temperature. The membranes were subsequently subjected to TBST washing three times for 10 min each time. The blots were developed with an enhanced chemiluminescence (Yeasen) and imaged using the chemiluminescence detection system. GAPDH served as a loading control.
7
Western Blot Analysis of Apoptosis Regulators
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Ovaries fragments or cells were lysed in RIPA buffer with protease inhibitor (Apexbio) and phosphatase inhibitor (Apexbio) for 10 mins and centrifuged at 12,000 g for 10 mins at 4 °C. The supernatant mixed with SDS‐PAGE sample buffer (GenStar) was denatured at 100 °C for 10 mins. Denatured protein was separated on 10–15% SDS‐PAGE gel and transferred to Polyvinylidene Fluoride membrane. Blocked with 5% non‐fat dry milk, the membranes were incubated with primary antibodies of GSDMD (Abcam, ab219800), IL1beta (Abcam, ab205924), caspase 1 (AdipoGen, AG‐20B‐0042‐C100), caspase 11 (AdipoGen, AG‐20B‐0060‐C100) and GAPDH (Affinity, AF7021) overnight at 4 °C and the corresponding secondary antibodies for 1 h at room temperature. The blots were imaged with ChemiDoc imaging system (Bio‐Rad).
8
Western Blot Analysis of Ion Channels
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Total protein was isolated from the BV2 cells using 100 μL precooled RIPA lysis buffer (Sigma‐Aldrich) with 1 μL protease inhibitor (ApexBio Technology) and centrifuged at 14,000 rpm at 4°C for 10 min. A BCA protein assay kit (Yeasen) was prepared for assessment of protein concentration. Protein (20 μg) was boiled in loading buffer, resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% nonfat milk, the membranes were incubated overnight with primary antibodies against Kir6.1 (ab241996, 1:1000; Abcam), SUR2A (Bio Excellence International Tech), Kir6.2 (ab79171, 1:1000; Abcam), SUR1 (ab217633, 1:1000; Abcam), and ACTIN (ab8227, 1:2000; Abcam) at 4°C. Then the membranes were incubated with secondary antibodies for 1 h at room temperature. Proteins were visualized by the enhanced chemiluminescence reagent (Yeasen). The immunoblot images were evaluated using ImageJ software.
9
Western Blot Analysis of Protein Expression
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In this study, cells were incubated in RIPA buffer (#P0013B; Beyotime, Shanghai, China) containing protease inhibitor (#K1007; APExBIO, Houston, America) and phosphatase inhibitor (#K1015; APExBIO) at 4°C. After 30 min, lysates were centrifuged at 12,000 rpm for 15 min at 4°C to obtain the supernatant. Proteins were quantified with a BCA assay kit (#P0010S, Beyotime), and 20 μg per lane were separated by SDS-PAGE and transferred to PVDF membranes (#IPVH00010, Millipore, Shanghai, America). Then, the membrane was incubated for 1 h at room temperature with HRP-conjugated antibody and developed using ECL reagent (#P0018S, Beyotime). Furthermore, antibodies against flag Tag (#8146, 1:1000), phospho-AKT (Ser473) (#13038, 1:1000), AKT (#4691, 1:3000), Bcl-2 (#15071, 1:1000), BAX (#5023, 1:1000) and cleaved Caspase-3 (#9661, 1:3000) were ordered from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA). Besides, cleaved PARP-1 (#ab32064, 1:1000) was purchased from Abcam (Abcam, Cambridge, UK), antibodies against β-actin (#YT0099, 1:3000) and HRP Goat anti-mouse IgG(H + L) (#RS0001,1:5000) from ImmunoWay (Jiangsu, China) and anti-rabbit IgG HRP-linked antibody (#7074, 1:5000) from Cell Signaling Technology.
10
Quantitative Protein Extraction and Western Blotting
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Total protein was extracted from cells using radio immunoprecipitation assay lysis (RIPA) buffer supplemented with protease inhibitor (1:100, Cat# K1007; ApexBio, Houston, United States) and the protein concentrations were measured using bicinchoninic acid (BCA) Protein Assay Kit (Cat# P0012S; Beyotime, Shanghai, China). The extraction and isolation of nuclear and cytoplasmic protein were performed according to the method adapted from Bayorh et al. (Bayorh et al., 2006 (link)) by using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China). An amount of 20 μg of total proteins was separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF, Thermo Fisher Scientific, United States) membranes. After being blocked with 5% non-fat milk in Tris-HCl buffer saline supplemented with 0.1% Tween-20 (TBST) at RT for 30 min, the membranes were incubated with primary antibodies (dilution of 1:1000) at 4°C overnight. Then, the membranes were washed with TBST for three times and incubated with secondary antibodies (dilution of 1:1000) for 1 h at RT. After being washed with TBST for three times, immunoreactive bands were visualized using electrochemiluminescence (ECL) reagent (Cat# 4AW011, 4A Biotech, Beijing, China) and quantified using ImageJ software.
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