Cells were solubilized in cold radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific, Inc.). The protein was extracted by centrifugation at 12,000 × g for 20 min at 4°C. The concentration of protein was determined using BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Proteins (50 µg/lane) were separated by 10% SDS-PAGE and subsequently transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.). The membrane was incubated with PBS containing 5% milk overnight at 4°C, which was then incubated with mouse anti-IGF-1R (1:200) and mouse anti-GAPDH (1:100, cat. no. ab8245, Abcam) monoclonal antibodies at room temperature for 3 h. After three washes with PBS, the membrane was incubated with a rabbit anti-mouse secondary antibody (1:10,000, cat. no. ab6728, Abcam) at room temperature for 1 h. Chemiluminescent detection was conducted using an Enhanced Chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.
Radioimmunoprecipitation lysis buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Radioimmunoprecipitation lysis buffer is a solution used to extract and solubilize proteins from cell samples for analysis. It is designed to disrupt cell membranes and release cellular contents, including proteins, while preserving their native state and interactions.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (3 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Rabbit anti mouse secondary antibody, by Abcam (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Gapdh, by Abcam (1 mentions)
Proteinase and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Ab93876, by Abcam (1 mentions)
Ab236639, by Abcam (1 mentions)
Ab2386, by Abcam (1 mentions)
Ab229126, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Enhanced chemiluminescence system, by Kodak (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Ab31013, by Abcam (1 mentions)
Ab184742, by Abcam (1 mentions)
18 protocols using radioimmunoprecipitation lysis buffer
1
IGF-1R Protein Detection and Quantification
2
Western Blot Analysis of FoxP3 Protein
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Cells were lysed in ice-cold radioimmunoprecipitation lysis buffer (no. 89900, Thermo Fisher Scientific, MA) for 30 min. Insoluble materials were removed, and the lysates were used for Western blotting or immunoprecipitated overnight with an antibody such as anti-FoxP3 antibody followed by incubation with protein G agarose beads at 4°C for 2 hours. The washed immunoprecipitates were boiled in SDS sample buffer, and the protein content was determined by the Bio-Rad protein assay kit (no. 5000002, Bio-Rad, Hercules, CA). Equal amounts (30 to 50 μg) were loaded onto 4 to 12% NuPage bis-tris precasting gels (SDS–polyacrylamide gel electrophoresis), transferred onto polyvinylidene difluoride membrane (Invitrogen), and immunoblotted. All blots were developed with the ECL immunodetection system (no. 426319, BioLegend, CA).
3
Western Blot Analysis of HSPA2 Protein
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Proteins were extracted using radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific, Inc.) and the protein content was determined using the Bicinchoninic Acid Protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of the protein (50 µg/lane) from lysates of PANC-1 cells were subjected to SDS-PAGE (10% gels) and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk for 60 min at room temperature, followed by incubation with primary antibodies against HSPA2 (cat. no. ab108416; 1:500; Abcam, Cambridge, MA, USA) and GAPDH (cat. no. AF0006; 1:1,000; Beyotime Institute of Biotechnology) overnight at 4°C. Membranes were then washed with 0.1% Tween-20 in PBS (PBST) three times at room temperature. Then, they were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (cat. no. sc-2004; 1:3,000; Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) at room temperature for 1 h. The proteins of interest were detected by the enhanced chemiluminescence detection system (Sea Biotech, Shanghai, China). Finally, the intensity of protein bands was detected using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). GAPDH served as the loading control.
4
Western Blotting of His-tagged Proteins
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Total protein was extracted using radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific, Inc.) and clarified by centrifugation at 4°C (12,000 × g for 20 min). Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 40 µg protein was separated by 10% SDS-PAGE (Bio-Rad Laboratories, Inc.), prior to being transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) and subsequently blocked with 5% dry skimmed milk in Tris-buffered saline for 1 h at room temperature. The membranes were incubated with an HRP-conjugated human anti-His antibody (cat. no. ab219465; 1:10,000; Abcam) overnight at 4°C, washed twice with PBS and subsequently incubated with HRP-conjugated goat anti-mouse IgG secondary antibodies for 1 h (cat. no. STAR137P; 1:3,000; Hercules) at room temperature. Protein bands were subsequently detected and visualized using a Super Signal WestPico chemiluminescent substrate (Thermo Fisher Scientific, Inc.).
5
Quantitative Western Blot Analysis
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rotein contents were extracted from cells using radio immunoprecipitation lysis buffer (#89901; Thermo Fisher Scientific, Waltham, USA) containing proteinase and phosphatase inhibitors (Thermo Fisher Scientific), and the concentration was determined using a BCA protein assay kit (#P0011; Beyotime, Shanghai, China). After separated by SDS-PAGE, protein samples were transferred to the PVDF membrane (#GVWP02500; Sigma-Aldrich, St. Louis, USA). GAPDH was used as a loading reference. The membrane was subsequently incubated with primary antibodies against TGM2 (#ab2386; 1:1000, Abcam, Cambridge, UK), alkaline phosphatase (ALP; #ab229126; 1:500, Abcam), osteocalcin (OCN; #ab93876; 1:1000, Abcam), and runt-related transcription factor 2 (RUNX2; #ab236639; 1:1000, Abcam) at 4 °C overnight followed by further incubation with secondary antibodies. An enhanced chemiluminescence system (Kodak, Rochester, USA) was applied to visualize the signals on the membrane. ImageJ software (NIH, Bethesda, USA) was used analyze the signal intensity of the blots.
6
Notch Signaling Pathway Analysis
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Macrophages were homogenized in radioimmunoprecipitation lysis buffer (Thermo Scientific). Protein samples were obtained by centrifugation for 10 min at 14,000 g at 4°C. Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes by the wet blotting procedure (100 V for 2 h at 4°C). Membranes were blocked with blocking buffer (5% skimmed milk in PBS containing 0.05% Tween-20) and incubated at 4°C overnight with the anti-Notch1 (1:1000, #4380; Cell Signaling Technology), anti-Notch4 (1:1000, #ab184742; Abcam), anti-Hes1 (1:1000, #11988S; Cell Signaling Technology), anti-TGF-β (1:1000, #ab31013; Abcam), anti-Smad3 (1:1000, #9523S; Cell Signaling Technology), or anti-GAPDH (1:1000, #2118S; Cell Signaling Technology) primary antibody. GAPDH was used as an internal control. After incubation of the membrane with an IRDye 800 anti-rabbit or IRDye 680 anti-mouse secondary antibody (LI-COR, USA) for 1 h at room temperature, densitometric analysis was performed using an Odyssey infrared imaging system (LI-COR).
7
Protein Expression Analysis by Western Blot
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The total protein of all cell lines was extracted using radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific, Inc.). The protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). A total of 40 µg protein was separated by 10% SDS-PAGE (Bio-Rad Laboratories, Inc.). The proteins were transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA) and were subsequently blocked with 5% non-fat dry milk in Tris-buffered saline for 1 h at room temperature. The membranes were incubated with the following primary antibodies: MIF (1:2,000), Twist1 (1:200), MMP-2 (1:200), MMP-9 (1:200) and α-tublin (1:3,000) overnight at 4°C, washed twice with phosphate-buffered saline (PBS) and were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories, Inc.) for 1 h at room temperature. The protein bands were subsequently detected with SuperSignal WestPico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.) and were visualized using a VersaDoc-MP Imaging system (Bio-Rad Laboratories, Inc.).
8
Hypoxia Modulates Lactate Metabolism
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HR-2 cells were grown in DMEM-S in 10-cm dishes for 24 h with 0.20 mM oxygen before they were moved to lower oxygen concentrations (0.04, 0.02, and 0.005 mM) for 48 h before harvest. At the time of harvest, cells were scraped into radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) and sonicated. Protein levels in the lysates were quantified by BCA (Thermo Fisher Scientific). Western blot analysis was conducted with an electrophoresis chamber and semi-dry turbo transfer system (BioRad, Hercules, CA, USA). The antibodies used were: LDHA: mAB #3582, P-LDHA pAB #8176, HSP90 (mAB #4877) (Cell Signaling Technology, Danvers, MA, USA); MCT-1 (pAB: T-19, sc-14917) and MCT-4 (pAB: H-90, sc-50329) (Santa Cruz Biotechnology, Dallas, TX, USA).
9
Western Blot Analysis of Protein Markers
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Total proteins from each well were harvested in ice-cold radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with phenylmethanesulfonyl fluoride for 1 h. The protein concentration was assessed with a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China), according to the manufacturer’s instructions. Equal protein content from each treatment was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were soaked in 5% skimmed milk as a blocking buffer for 1 h, then washed in Trisbuffered saline Tween20 three times at room temperature. The membranes were incubated with primary monoclonal antibodies against GRP78, CHOP, phospho-eIF2α, AIF, bcl-2, bax, cytochrome-C, Nrf2, NQO-1, HO-1, and caspase3/cleaved-caspase3 overnight at 4°C followed by hybridization with horseradish peroxidase-conjugated secondary antibody. Relative protein levels were calculated based on β-actin as the loading control. Signal detection was visualized by enhanced chemiluminescence.
10
Protein Expression Analysis via Western Blot
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Radio immunoprecipitation lysis buffer (Thermo Fisher Scientific, Ma, USA) was applied to extract the total protein of the cells after transfection. The total protein concentration was determined using a bicinchoninic acid protein detection kit (Thermo Fisher Scientific). Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio‐Rad Laboratories). After blocking the membranes with the primary antibody (FLAG, Cell signaling Technology USA Cat. No. 8146; dilution of 1:1000) at 4°C overnight, they were then incubated with secondary antibody (anti‐mouse IgG, HRP‐linked antibody, CST USA Cat. No. 7076; dilution of 1:5000) at 27°C for 2 h. The target bands were finally detected using a chemiluminescent substrate (Perkin Elmer). Tanon‐5200Multi (Tanon) was used for exposure imaging. Protein expression was analyzed using the ImageQuant (LAS‐4000, FujiFilm) software. Bands were quantified using ImageJ software.
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