Zinc sulfate, rapamycin, trisaminomethane (Tris), radioimmunoprecipitation assay (RIPA), sodium dodecyl sulfate (SDS) buffer, and protease inhibitor cocktail were obtained from Sigma Aldrich Co. (St. Louis, MO, USA). A Bradford kit was purchased from Bio-Rad (CA, USA). For the primary antibodies employed in the present study, please refer to Supplementary Table 1
Radio immunoprecipitation assay ripa
Manufactured by Merck Group
Sourced in United States
The Radio-Immunoprecipitation Assay (RIPA) is a laboratory technique used for the detection and quantification of specific proteins in a sample. It combines immunoprecipitation and radioactive labeling to isolate and measure target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Sox2 ab171380, by Abcam (3 mentions)
Tubulin 2148, by Cell Signaling Technology (3 mentions)
β actin, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (2 mentions)
Rapamycin, by Merck Group (1 mentions)
Bradford kit, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Zinc sulfate, by Merck Group (1 mentions)
Skim milk, by Merck Group (1 mentions)
Biotinylated secondary antibody, by Abcam (1 mentions)
Bicinchoninic acid solution, by Merck Group (1 mentions)
Scopolamine hydrobromide, by Merck Group (1 mentions)
Nerve growth factor (ngf), by Abcam (1 mentions)
Cresyl violet acetate, by Merck Group (1 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (1 mentions)
Doublecortin dcx, by Abcam (1 mentions)
9 amino 1 2 3 4 tetrahydroacridine hydrochloride hydrate, by Merck Group (1 mentions)
Avidin biotin peroxidase complex, by Abcam (1 mentions)
Phospho creb, by Abcam (1 mentions)
12 protocols using radio immunoprecipitation assay ripa
1
Protein Analysis Using RIPA Buffer
2
Neurobiological Assays in Cognitive Studies
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The following reagents were obtained from Sigma (St. Louis, MO, USA); acetic acid, bicinchoninic acid solution, bovine serum albumin, copper(II) sulfate pentahydrate, Cresyl violet acetate, 3,3′-diamino-benzidine (DAB), radioimmunoprecipitation assay (RIPA), scopolamine hydrobromide, skim milk, and 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate (THA). The CREB, phospho-CREB, NGF, BDNF, beta-actin, and secondary horseradish peroxidase (HRP)-conjugated antibodies used for western blotting were obtained from Abcam (Cambridge, MA) and Santa Cruz Biotechnology (Santa Cruz, CA); the doublecortin (DCX), biotinylated secondary antibodies, and avidin-biotin peroxidase complex used for immunohistochemical staining were obtained from Abcam (Cambridge, MA), Santa Cruz Biotechnology (Santa Cruz, CA) and Vector Laboratories (Burlingame, CA).
3
Proteomic Analysis of Sperm Antioxidant Effects
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Proteomic profiling of sperm samples was performed using liquid chromatography-tandem mass spectrometry before and after antioxidant treatment. The samples were analyzed in compliance with the Minimum Information about a Proteomics Experiment (MIAPE) guidelines of the Human Proteome Organization’s Proteomics Standards Initiative (HUPO-PSI) for reporting proteomics studies [25 (link)]. The sperm pellet was washed four times with phosphate buffered saline (PBS; Irvine Scientific, Santa Ana, CA, USA) and centrifuged at 4000× g for 10 min, at 4 °C. Radio-immunoprecipitation assay (RIPA; Sigma-Aldrich, St. Louis, MO, USA) buffer supplemented with Protease Inhibitor Cocktail, cOmpleteTM ULTRA Tablets, EDTA-free (Roche, Mannheim, Germany) was added to the sperm pellet (100 µL RIPA/106 sperm) and left overnight at 4 °C for cell lysis. The samples were centrifuged at 10,000× g for 30 min at 4 °C and the supernatant was then transferred to a new centrifuge tube. Protein quantification in the fractions was performed using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
4
Quantification and Analysis of Phosphorylated EGFR
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The isolation of protein was carried out following lysis of tumor tissues in Radio-immunoprecipitation assay (RIPA, Sigma) buffer in the presence of protease and phosphatase inhibitors (Sigma) and quantified by modified Lowry method using Bio-Rad’s DC Protein Assay Reagent. Protein samples were run under denaturing conditions in SDS-PAGE in the presence of a standard molecular weight marker. Proteins from the gel were transferred onto nitrocellulose membrane and incubated with primary (p-EGFR, PY1068, rabbit polyclonal, dilution 1:500, Cell Signaling Technology; EGFR, rabbit polyclonal, dilution 1:1000, Cell Signaling Technology; and αTubulin, Imgenex) and secondary antibodies (Anti-Rabbit-HRP conjugate, Cell Signaling Technology), and detected by a ECL documentation system (GE Healthcare).
5
Western Blot Analysis of Protein Expression
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The total proteins were lysed in ice-cold Radio-Immunoprecipitation Assay (RIPA, Sigma) lysis buffer and separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene difluoride membrane (Millipore, USA). After blocking with 5% fat-free milk, the proteins were incubated with antibodies for Annexin A1, TSG101, ARF6, β-actin, p38, p-p38(Abcam, UK) overnight at 4 ℃. Subsequently, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies (Thermo Fisher Scientific, USA). The bands were visualized using the ECL detection system (Millipore, USA). Quantity One software (Bio-Rad) was used to detect the band intensity.
6
Isolation and Characterization of CD45+ Microvesicles
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Microvesicles were separated as described above and resuspended in PBS+BSA 1%. Anti-CD45 antibody (Santa Cruz Biotechnology, United States) was coupled to Dynabeads (Invitrogen, United States) and then incubated with MVs overnight at 4°C. Proteins retained (immunoprecipitated, IP) on the beads were recovered by adding cold Radio-Immunoprecipitation Assay (RIPA) buffer containing phosphatase and protease inhibitors (Sigma-Aldrich, Italy) and Laemmli buffer 2× and boiled at 95°C for 5 min to obtain CD45+ MVs. Immunodepleted fraction (I-) and input (starting material of MVs) were lysed in cold RIPA buffer, mixed with Laemmli buffer 2× and denatured at 95°C for 10 min.
7
Quantifying Transcytosis in Brain Microvascular Endothelial Cells
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Dextran (Alexa-Fluor® 488, 10 kDa, 10 μM; Sigma) was used to measure the amount of transcytotic activity in the BMECs. Transwells were removed from co-culture and replaced in EC ± medium in the basolateral chamber. Dextran, diluted in EC ± was added to the apical side of the transwells on a rotating platform at 37 °C or 4 °C for 2 h. To determine the rate of transcytosis, media was collected from the basolateral chamber and read on a fluorescent plate reader. BMECs were then rinsed twice in phosphate buffer solution (PBS; Sigma) and lysed with radioimmunoprecipitation assay (RIPA; Sigma). After trituration the lysate was collected and quantified using a fluorescent plate reader, this value is the Dextran accumulated at this time point. Both transcytosis and accumulation values were normalized to protein content as determined by a bicinchoninic acid assay (BCA; Sigma).
8
PD-L1 Protein Extraction and Analysis
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Proteins from cells and tissues were extracted using Radio-Immunoprecipitation Assay (RIPA; Sigma, United States) buffer with protease inhibitor Phenylmethanesulfonyl Fluoride (PMSF; Sigma) added, then incubated on ice for 20 min; cell lysates were centrifuged at 10,000 g for 5 min at 4°C, and then supernatants obtained were incubated at 99°C for 10 min to denature the proteins. Western blot analysis was conducted following standard protocol. The following antibodies were used: rat anti-mouse PD-L1 (1:1000, R&D, United States, MAB1019) and mouse anti-Actin (1:5000, Sigma, A2228).
9
Western Blot Analysis of Stem Cell Markers
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YAP1(#4912), LATS1(#9153), p-YAP1(#4911), Vimentin(#12020), Snail(#3879),and Tubulin(#2148) antibodies were all from Cell Signaling Technology (CST, Danvers, MA). OCT4 (ab19875), CD133 (ab19898) and SOX2 (ab171380) were from Abcam. Protein was obtained from cells by using Radio-Immunoprecipitation Assay (RIPA) (Sigma-Aldrich). Equal quantities of protein lysates were loaded on the Sodium dodecyl sulfate–Polyacrylamide Gel Electrophoresis gels (SDS-PAGE gels), and gel electrophoresis was applied to transfer the protein onto a nitrocellulose membrane. The protein strips were ultimately detected by enhanced chemiluminescence (Thermo Fisher Scientific) and captured using the Odyssey Infrared Imaging Analysis Software.
10
Molecular Profiling of LSCC Cancer Stem Cells
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For research use, the LSCC samples were collected from the Department of Oncology, Shanghai Chest Hospital. Written consent forms were obtained from every LSCC patient. The Institute Research Ethics Committee of Shanghai Chest Hospital approved all protocols.
Western Blotting YAP1(#4912), LATS1(#9153), p-YAP1(#4911), Vimentin(#12020), Snail(#3879) and Tubulin(#2148) antibodies were all from Cell Signaling Technology (CST, Danvers, MA). OCT4 (ab19875), CD133 (ab19898) and SOX2 (ab171380) were from Abcam. Protein was obtained from cells by using Radio-Immunoprecipitation Assay (RIPA) (Sigma-Aldrich). Equal quantities of protein lysates were loaded on the Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis gels (SDS-PAGE gels), and gel electrophoresis was applied to transfer the protein onto a nitrocellulose membrane. The protein strips were ultimately detected by enhanced chemiluminescence (Thermo Fisher Scienti c) and captured using the Odyssey Infrared Imaging Analysis Software.
Western Blotting YAP1(#4912), LATS1(#9153), p-YAP1(#4911), Vimentin(#12020), Snail(#3879) and Tubulin(#2148) antibodies were all from Cell Signaling Technology (CST, Danvers, MA). OCT4 (ab19875), CD133 (ab19898) and SOX2 (ab171380) were from Abcam. Protein was obtained from cells by using Radio-Immunoprecipitation Assay (RIPA) (Sigma-Aldrich). Equal quantities of protein lysates were loaded on the Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis gels (SDS-PAGE gels), and gel electrophoresis was applied to transfer the protein onto a nitrocellulose membrane. The protein strips were ultimately detected by enhanced chemiluminescence (Thermo Fisher Scienti c) and captured using the Odyssey Infrared Imaging Analysis Software.
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