Total protein was extracted from Treg cells using radioimmunoprecipitation assay buffer (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 50 µg protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Subsequently, the membrane was blocked with 5% skim milk at room temperature for 1 h and incubated with primary antibodies against MMP-9 (cat. no. ab73734; 1:500; Abcam, Cambridge, MA, USA) and GAPDH (cat. no. ab9485; 1:500; Abcam) at 4°C overnight. Following washing with PBST three times, the membrane was incubated with a horseradish peroxidase conjugated anti-rabbit IgG antibody (cat. no. ab191866; 1:500; Abcam) for 1 h at 37°C. Finally, the protein expression was detected by enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.). Chemiluminescent detection was performed using Bio-Rad ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Radioimmunoprecipitation assay buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Switzerland, Japan, Germany
Radioimmunoprecipitation assay buffer is a laboratory reagent used in the process of radioimmunoprecipitation assay (RIPA). It is designed to lyse cells and solubilize proteins for subsequent analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (27 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (19 mentions)
Polyvinylidene difluoride membrane, by Merck Group (14 mentions)
Protease inhibitor cocktail, by Roche (13 mentions)
β actin, by Cell Signaling Technology (11 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (11 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (10 mentions)
Polyvinylidene fluoride membrane, by Merck Group (9 mentions)
Tween 20, by Merck Group (9 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (9 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (9 mentions)
Ab9485, by Abcam (8 mentions)
Pvdf membrane, by Thermo Fisher Scientific (8 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (8 mentions)
Image pro plus software 6, by Media Cybernetics (7 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Quantity one software, by Bio-Rad (6 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Protease inhibitor, by Thermo Fisher Scientific (6 mentions)
278 protocols using radioimmunoprecipitation assay buffer
1
Western Blot Analysis of MMP-9 in Treg Cells
2
Western Blot Analysis of Signaling Proteins
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Cells were washed in phosphate-buffered saline (PBS) and lysed using radioimmunoprecipitation assay buffer (Invitrogen, Carlsbad, CA) supplemented with a protease inhibitor cocktail (Roche, Pleasanton, CA, USA). The protein concentration was evaluated using a bicinchoninic acid protein assay kit (Bioswamp, PAB180007, Wuhan, China). Equivalent amounts of proteins (30 μg) from each sample were subjected to sodium dodecyl-polyacrylamide electrophoresis, transferred to a polyvinylidene fluoride membrane, blocked in 5% fat-free milk for 2 hours at room temperature, and incubated with the following primary antibodies: PTEN (ab32199, 1:10,000, abcam), PI3K (ab191606, 1:10,000, abcam), p-PI3K (ab182651, 1:10,000, abcam), mTOR (ab32028, 1:10,000, abcam), p-mTOR (ab109268, 1:10,000, abcam), GLUT1 (ab652, 1:10,000, abcam), LDHB (ab85319, 1:10,000, abcam), HK2 (ab209847, 1:10,000, abcam), PKM2 (ab150377, 1:10,000, abcam), or GAPDH (PAB36264, 1:10,000, Bioswamp). Then, the membranes were washed with Tris-buffered saline and incubated with goat anti-rabbit IgG secondary antibody (SAB43711, 1:10,000, Bioswamp) for 2 h at room temperature. An enhanced chemiluminescence kit (Pierce) was used to detect specific bands and autoradiograms were quantified by densitometry (Quantity One software, Bio-Rad, Hercules, CA, USA) using GAPDH as a control. For each group, the quantification was triplicated.
3
Protein extraction and Western blotting
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Drug treated cells were lysed for total protein extraction using radioimmunoprecipitation assay buffer (Invitrogen). Protein concentrations were measured using QuantiPro BCA Assay kit (Invitrogen). Equal amount of proteins was loaded onto denaturing sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) and resolved via electrophoresis. Protein were then transferred onto polyvinylidene fluoride membranes (Bio-Rad), followed by WB analysis using standard protocol. The blots were cut prior to hybridisation with antibodies during blotting. All antibodies were purchased from Abcam. Signal was developed using enhanced chemiluminescent reagent (Pierce).
4
Quantitative Western Blot Analysis
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Western blot assay was performed to determine the protein expression levels in each group. The cells were lysed in cold radioimmunoprecipitation assay buffer (Invitrogen Life Technologies, Carlsbad, CA, USA). A BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the protein concentrations, according to the manufacturer’s instruction. Subsequently, the proteins were separated on a 10% sodium dodecyl sulphate-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% fat dry milk in PBS for 4 h. Subsequently, the PVDF membrane was incubated with specific primary antibodies (mouse anti-cyclin D1, mouse anti-PCNA, mouse anti-CDK4, mouse anti-smooth muscle-α-actin, mouse anti-smoothelin, mouse anti-desmin, mouse anti-phospho-Akt, mouse-anti-Akt, anti-phospho-ERK, mouse-anti-ERK, and mouse anti-glyceraldehyde 3-phosphate dehydrogenase antibodies; all from Abcam, Cambridge, UK) for 3 h. After washing three times with PBS (5 min each time), the PVDF membrane was incubated with a rabbit anti-mouse secondary antibody (Abcam). Next, after washing three times with PBS (5 min each time), an enhanced chemiluminescence western blotting kit (Thermo Fisher Scientific) was used to detect the immune complexes present on the PVDF membrane.
5
Protein Expression Analysis Protocol
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Protein expression was detected after extraction of proteins in tissues and cells using radio immunoprecipitation assay buffer (Invitrogen). After being separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane (Millipore Corp, Billerica, MA, United States). After being sealed with 5% skim milk for 2 h, the membrane was incubated with primary antibodies at 4°C overnight, followed by 2-h incubation with corresponding rabbit secondary antibody (1:4,000, Abcam, Cambridge, United States) at 37°C. Immunoreactive proteins were visualized using enhanced chemiluminescence (Millipore). The primary antibodies included α-SMA (1:1,000; Abcam), fibronectin (1:1,000; Abcam), collagen IV (1:1,000; Abcam), p53-upregulated modulator of apoptosis (PUMA, 1:1,000, #24633, CST), apoptotic protease activating factor 1 (Apaf1, 1:1,000, ab2001, Abcam), B-cell CLL/lymphoma 2 (Bcl-2, #3498, CST), TGF-β1 (1:1,000, #MA5-15065, Thermo Fisher), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1,000; Abcam).
6
Chondrocyte Protein Expression Analysis
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HACs were lysed in radioimmunoprecipitation assay buffer (Invitrogen) supplemented with protease inhibitor cocktail (Sigma–Aldrich). After lysis, total proteins were separated in a NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen) and transferred to polyvinylidene difluoride nylon membranes (Sigma–Aldrich). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and incubated overnight at 4 °C with primary antibodies against COX-2 (sc-7951; Santa Cruz Biotechnology, CA, USA), Col10a1 (bs0554 R; Bioss, MA, USA), PTHrP (TA334682; OriGene Technology, MD, USA) and GAPDH (MA5-15738; Thermo Scientific, MA, USA). Specific bands were detected with a horseradish peroxidase-conjugated secondary antibody and were read using an enhanced chemiluminescence western blot system from Pierce (Rockford, IL, USA).
7
iPSC Protein Extraction and Western Blot
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Proteins were extracted from iPSCs and TSLCs were by radioimmunoprecipitation assay buffer (Invitrogen) containing protease inhibitor and phosphatase inhibitors (both from Invitrogen). Thirty micrograms of total protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to 0.45 µm nitrocellulose membranes (Millipore). Blots were blocked with 5% skimmed milk in Tris-buffered saline for 1 hour, incubated with primary antibodies at 4°C overnight, and then incubated with horseradish peroxidase-coupled secondary antibodies at 37°C for 1 hour. The primary antibodies used as follow: NME4 (1:1,000; Novus Biological, Centennial, CO, USA), p53 (1:1,000; Santa Cruz Biotechnology), thioredoxin (TRX) (1:250; Abcam) and β-actin (1:1,000; Santa Cruz Biotechnology).
8
Extracellular Vesicle Protein Analysis
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HACs were lysed in radioimmunoprecipitation assay buffer (Invitrogen) supplemented with protease inhibitor cocktails (mixtures) (Sigma-Aldrich). Following lysis, proteins were separated by a NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen) and transferred to polyvinylidene difluoride nylon membranes (Sigma-Aldrich). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and incubated overnight at 4 °C with primary antibodies against CD9 (cat. no. 602321lg, Proteintech Group, Inc., Rosemont, IL, USA; 1:1000), CD63 (256821-AP, Proteintech Group, Inc.; 1:1000), CD81 (66866-1lg, Proteintech), ALIX (cat. no. 12422-1-AP, Proteintech Group, Inc.; 1:1000), TSG101 (282831-AP, Proteintech Group, Inc.; 1:1000), and α-tubulin (cat. no. 66009-1-lg; Proteintech Group, Inc.; 1:1000). Specific bands were detected by a horseradish peroxidase–conjugated secondary antibody and were read by an enhanced chemiluminescence Western blot system from Pierce (Rockford, IL, USA).
9
Western Blot Analysis of Apoptosis Markers
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Proteins were extracted from the cells using radioimmunoprecipitation assay buffer (Invitrogen), and then, the sample concentration was assessed using a bicinchoninic acid protein quantification kit (Thermo Fisher Scientific). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene fluoride membranes (Millipore, Temecula, CA), blocked with 5% skim milk, and then incubated with the following primary antibodies: anti-YY1 (#46395, Cell Signaling Technology, Danvers, MA, USA, 1:1000), anti-PARP (#9542, Cell Signaling Technology, 1:1000), anti-Caspase-3 (#9662, Cell Signaling Technology, 1:1000), and anti-GAPDH (#5174, Cell Signaling Technology, 1:1000). Next, the membranes were incubated with secondary antibodies. Protein signals were observed using enhanced chemiluminescence (Bio-Rad), with GAPDH as an internal control (Olympus, Tokyo, Japan).
10
Protein Expression Profiling by Western Blot
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Protein extracts were obtained by radioimmunoprecipitation assay buffer (Invitrogen, Waltham, MA USA) containing 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS. Equivalent amounts of protein extracts were separated using SDS-PAGE and transferred to polyvinylidene difluoride membrane (Sigma-Aldrich, St. Louis, MO). After blocking the membranes with 8% fat-free milk (in TBS with 0.1% Tween 20) for 1 h at 37 °C, primary antibodies were applied as 1:1000 including anti-RER1 (Sigma, St. Louis, MO), anti-E-cadherin (24E10, Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (clone GC-4, Sigma, St. Louis, MO), anti-vimentin (RV202, Abcam, Cambridge, UK, USA), anti-snail (ab180714, Abcam), anti-claudin 1(ab15098, Abcam), anti-Sox2 (AB5603, Millipore, Billerica, MA, USA), anti-Bmi1 (ab38295; Abcam), anti-Lin28 (ab46020, Abcam), anti-Nanog (ab62734, Abcam), HIF-1α (ab65979, Abcam) and anti-actin (Sigma, A2066). Actin was used as a loading control. HRP-conjugated secondary goat anti-rabbit or goat anti-mouse (Santa Cruz, Dallas, TX, USA) antibodies were applied to the membranes, which were then developed using a ECL chemiluminescence kit (Pierce, Waltham, MA). After exposure, films were scanned and quantified using the NIH Image J software.
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