The cultured EPCs were cleaned and hatched in radioimmunoprecipitation assay buffer containing inhibitors of proteases (Roche, China). For protein level determination, we used a bicinchoninic acid (BCA) kit (Thermo Scientific, Waltham, USA). Proteins were electrophoretically separated by 10% sodium dodecyl sulfate-polyacrylamide gel and were transferred to polyvinylidene difluoride (Millipore, Bedford) membranes. The membrane was then blotted with 5% bovine serum albumin and overnight with antibodies against p-p38 total p38, p-Akt, total Akt and β-actin (Cell Signaling Technology) at dilutions indicated by the manufacturer. Anti-rabbit or anti-mouse secondary antibodies and an ECL chemiluminescence detection system (Pierce Biotechnology Inc, Rockford, IL) were applied to scan and semi-quantitatively analyze the proteins according to the manufacturer’s instructions.
Radioimmunoprecipitation assay buffer
Manufactured by Roche
Sourced in Switzerland
Radioimmunoprecipitation assay buffer is a laboratory reagent used in the process of radioimmunoprecipitation assay (RIPA), a technique used to extract and analyze proteins from cell or tissue samples. The buffer's core function is to solubilize and denature proteins, allowing for their subsequent immunoprecipitation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (6 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
P iκbα, by Cell Signaling Technology (2 mentions)
Blc 2, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride, by Merck Group (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Gapdh antibody, by Abcam (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
Supersignal west dura extended duration substrate kit, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by ZSGB-BIO (1 mentions)
Anti green fluorescent protein, by Abcam (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Aggrecan, by Abcam (1 mentions)
13 protocols using radioimmunoprecipitation assay buffer
1
Western Blot Analysis of Cultured EPCs
2
Evaluation of TGF-β1/Smad Signaling in Kidney Fibrosis
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Collected kidney tissues or RMCs were lysed in radioimmunoprecipitation assay buffer (Roche Diagnostics) using manufacturer's protocol. Protein concentration was measured using the Micro BCA protein assay kit (Youdi Bio-technology Co., Ltd, China). Total proteins (50 µg) were loaded into each lane and 12% SDS-PAGE was performed. Cells were then transferred to PVDF membranes (Thermo Fisher Scientific, Inc.). The PVDF membranes were blocked with 6% non-fat dry milk at 37°C for 1 h. Immunoblotting was performed using anti-TGF-β1 (1:200; cat. no. ab92486), anti-Smad3 (1:1,000; cat. no. ab40854), anti-Smad7 (1:500; cat. no. ab227309), anti-collagen type 1 (col1; 1:500; cat. no. ab6308), anti-α-smooth muscle actin (anti-α-SMA; 1:1,000; cat. no. ab32575) and anti-GAPDH (1:1,000; cat. no. ab8245) at 37°C for 2 h, followed by incubation with anti-rabbit/mouse horseradish peroxidase-conjugated IgG secondary antibodies (1:5,000; cat. nos. ab6721 and ab190475, respectively) at 37°C for 1 h. All antibodies were from Abcam. Antibody incubation was followed the detection of immunoblots and visualization using enhanced chemiluminescence Western Blotting Substrate (Pierce; Thermo Fisher Scientific, Inc.; cat. no. 32106). GAPDH levels were used for normalization. Protein bands were scanned and quantified using a ChemiDoc MP Image analysis system (cat. no. 170-8280; Bio-Rad Laboratories, Inc.).
3
Mitochondrial Protein Expression Analysis
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Tissues were homogenized in radioimmunoprecipitation assay buffer (Roche Applied Science), and protein concentration was determined by bicinchoninic acid protein assay (Pierce). An even concentration of protein lysates was separated in SDS-PAGE gels, transferred to nitrocellulose membranes (Millipore), and incubated with Ucp-1, Cytochrome C, NADH dehydrogenase Fe-S protein 3 (NDUFS3), ATP5a and GAPDH antibodies (Abcam). Quantification of signals was performed using a Gel Doc XR system (Bio-Rad).
4
Protein Expression Analysis in SGC-7901 Cells
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SGC-7901 cells (5×105) were seeded in 6-well plates and transfected with small interfering RNAs (siRNAs). After 24 h, the cells were harvested and lysed in radioimmunoprecipitation assay buffer (Roche Diagnostics, Basel, Switzerland). Total proteins were measured using a BCA kit (Thermo Fisher Scientific, Inc.), and 20 µg was loaded for 12% SDS-PAGE and separated, followed by transferred onto polyvinylidene difluoride (PVDF) membranes and blocked in PBS containing 3% bovine serum albumin at room temperature for 1 h. Anti-F cyclin D1 rabbit (dilution, 1:500; cat. no. 2978), anti-cyclin E1 mouse (dilution, 1:500; cat. no. 4129) and anti-GAPDH rabbit (dilution, 1:1,000; cat. no. 5174) antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) were incubated at 4°C overnight. The goat-anti-rabbit (cat. no. ZB-2301) or goat-anti-mouse (cat. no. ZB-2305) HRP-conjugated secondary antibodies (1:10,000; ZSGB-BIO, Beijing, China) were incubated at room temperature for 1 h. Finally, the PVDF membranes were visualized using SuperSignal West Dura Extended Duration Substrate kit (Thermo Fisher Scientific, Inc.).
5
Western Blot and Co-Immunoprecipitation Assays
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Radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (Roche, Basel, Switzerland) was used to lyse spinal cord or brain samples for western blot assays, while a non-denaturing lysis buffer [1% NP40, 5 mM ethylenediaminetetraacetic acid, 50 mM Tris-HCl (pH 8), 150 mM NaCl] containing the same inhibitors was used to lyse samples for co-immunoprecipitation. Samples were then separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4–12% gels (Cat# V900859; Sigma-Aldrich, St. Louis, MO, USA). Protein bands were detected using enhanced chemiluminescence-Prime (Amersham, Buckinghamshire, UK) reagent. Densitometric analyses were conducted with Image Lab software (Bio-Rad, Hercules, CA, USA). Antibodies used for these analyses included anti-glyceraldehyde-3-phosphate dehydrogenase (mouse; 1:5000; Cat# GB12002; Servicebio), anti-CRMP3 (Cat# bs-11821R; 1:200; Bioss), anti-spastin (Cat# ab77144; 1:1000; Abcam), anti-horseradish peroxidase-conjugated goat anti-rabbit (1:5000; Cat# AS014; Abclonal Technology, Woburn, MA, USA), anti-horseradish peroxidase-conjugated goat anti-mouse (1:5000; Cat# AS003; Abclonal Technology), and anti-green fluorescent protein (GFP; 1:2000; Cat# ab290; Abcam). The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated at room temperature for one hour.
6
Western Blot Analysis of Cell Markers
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CHON-001 cell lysates were washed with PBS and homogenized in radioimmunoprecipitation assay buffer (Roche Diagnostics, Basel, Switzerland). Proteins (30 µg per lane) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were blocked with 5% milk in Tris buffered saline with 0.1% Tween-20 for 2 h at room temperature. Next, they were incubated with primary antibodies against β-actin (cat no. 4970; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CXCL12 (cat no. 3530; 1:1,000; Cell Signaling Technology, Inc.), type I collagen (cat no. ab34710; 1:1,000; Abcam) and aggrecan (cat no. ab36861; 1:1,000; Abcam) at 4°C overnight. Membranes were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (cat no. 7074; 1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Bands were visualized by SuperSignal® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.). Band density was normalized to β-actin.
7
Western Blot Analysis of Inflammatory Signaling
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Cells were lysed in ice-cold radioimmunoprecipitation assay buffer (Roche, Basel, Switzerland) supplemented with protease inhibitors. Protein concentrations were determined using dioctanoic acid assays (Thermo Fisher Scientific). Equal volumes of proteins were separated on 10% sodium lauryl sulfate-polyacrylamide gels. Separated proteins were transferred to polyvinylidene fluoride membranes and blocked in 1.5% skimmed milk in Tris buffered saline containing Tween 20 (TBST). Membranes were probed with primary antibodies at 4 °C overnight and washed in TBST. The membranes were then probed with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 2 h at room temperature. ECL kits (Thermo Fisher Scientific) were used to determine band intensities on the membranes as per the manufacturer’s recommendations. The primary antibodies were TLR4, p-p65, p65, p-IκBα, IκBα, Blc-2, Bax, and caspase-3 (Cell Signaling, Danfoss, Mass.) GAPDH (Santa Cruz, California) was also probed as a loading control.
8
Western Blot Analysis of HACD2, FBXO8, and TM9SF3
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Total protein was homogenized with radioimmunoprecipitation assay buffer (Roche Diagnostics GmbH, Mannheim, Germany), separated by electrophoresis using 8–10% sodium dodecyl sulfate gels, and transferred to polyvinylidene fluoride membranes (EMD Millipore Corporation, Billerica, MA, USA), which were blocked with 10% skim milk for 2 h and then probed with antibodies against HACD2 (bs-4429R; Bioss, Inc., Woburn, MA, USA), FBXO8 (bs-16053R; Bioss, Inc.), TM9SF3 (bs-19944R; Bioss, Inc.), and β-actin (8227; Abcam, Cambridge, MA, USA) and visualized with an enhanced chemiluminescence kit (WBKLS0050; EMD Millipore Corporation). The bands were imaged using a chemiluminescence imaging system (Syngene, Frederic, MD, USA).
9
Western Blot Analysis of Cell Signaling
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The cell lysates were homogenized in radio-immunoprecipitation assay buffer (Roche, CA, USA). The proteins were separated by 12% sodium dodecyl sulfate polyacrylamide (SDS) gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, USA), which were subsequently incubated with primary antibodies overnight at 4 °C. Following incubation with the specific HRP-conjugated Goat Anti-Rabbit IgG, chemiluminescence signal was detected using BeyoECL Plus (Beyotime, China). Antibodies against Cyclin D1 (# WL01435a), CDK2 (# WL01543), CDK4 (# WL01711), Bax (# WL01637), pro-caspase 3 (# WL02117), cleaved-caspase 3 (# WL02117), pro-caspase 9 (# WL03421), and cleaved-caspase 9 (# WL03421) were purchased from Wanleibio (Shenyang, Liaoning, China). Antibody against Titin/CMD1G (# bs-9861R) was purchased from Bioss (Beijing, China). Antibody against β-actin (# 70-ab36861–050) and HRP-conjugated Goat Anti-Rabbit IgG (# 70-GAR007) were purchased from Liankebio (Hangzhou, Zhejiang, China). Protein expression was normalized to the β-actin level.
10
Western Blot Protein Analysis
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Radioimmunoprecipitation assay buffer (Roche Diagnostics, Basel, Switzerland) was added to lyse the cells. The supernatant was centrifuged at 12,000 × g for 10 min to obtain the total protein solution. A bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, United States) was used to determine the protein concentration; each 15 μL sample contained 40 μg of protein. After electrophoresis, the proteins in the gel were transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States), which were incubated with primary antibodies (1:1,000) overnight at 4°C. Secondary antibodies (1:2,000) were added to the membranes and incubated for 1 h the next day. Finally, an enhanced chemiluminescence kit (Applygen Inst. Biotech, Beijing, China) was used to detect the protein bands.
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