Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Bicinchoninic acid (BCA) protein assay kit and Imperial Protein stain was from Pierce (Thermo Scientific, Rockford, IL, USA). Mini-protean TGX 4-20% polyacrylamide gels and Transfer-Blot Turbo Transfer Pack were from Bio-Rad (Hercules, CA, USA). PVDF membranes were from Millipore (Billerica, MA, USA). The antibodies used for Western blotting were: mouse anti-Tsg101 (BD Biosciences, Heidelberg, Germany); rabbit anti-CD9 (Abcam, Cambridge, UK); mouse anti-CD81 (Ancell Corporation, Bayport, MN, USA), rabbit anti-uromodulin (St. Cruz Biotechnology Inc., Dallas, TX, USA). HRP-conjugated secondary antibodies were from Jackson Immunoresearch (West Grove, PA, USA). The antibodies used for immuno-electron microscopy were: mouse anti-CD63 (H5C6) (DSHB, Iowa city, IA, USA) and rabbit-anti-mouse (DACO, Glostrup, Denmark). Protein A-gold conjugates (10 nm) were purchased from Cell Microscopy Center (Utrecht, Netherlands).
Rabbit anti cd9
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Rabbit anti-CD9 is a primary antibody that recognizes the CD9 antigen, a tetraspanin membrane protein. This antibody can be used for the detection and analysis of CD9 expression in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti tsg101, by Abcam (5 mentions)
Rabbit anti cd63, by Abcam (4 mentions)
Rabbit anti cd81, by Abcam (3 mentions)
Mouse anti cd63, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Rabbit anti gapdh, by Abcam (2 mentions)
Pierce enhanced chemiluminescence western blotting substrate, by Merck Group (2 mentions)
Rabbit anti cd63, by Wanlei (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx 4 20 polyacrylamide gels, by Bio-Rad (1 mentions)
Transfer blot turbo transfer pack, by Bio-Rad (1 mentions)
Mouse anti tsg101, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rabbit anti smad5, by Proteintech (1 mentions)
Anti gapdh antibody, by Proteintech (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Rabbit anti pgc 1α, by Abcam (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
16 protocols using rabbit anti cd9
1
Extracellular Vesicle Protein Characterization
2
Western Blot Analysis of Exosomal Markers
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Protein samples were boiled in SDS/β-mercaptoethanol sample buffer, and 20 μg of protein from each sample was loaded on a gel. The antibodies used for western blotting were rabbit anti-CD9 (Abcam), rabbit anti-CD63 (Abcam) and rabbit anti-SMAD5 (Proteintech). Anti-GAPDH antibody (Proteintech) was used to detect GAPDH, which served as a loading control.
3
Western Blot Analysis of Exosomal Markers
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Exosomes and cells lysed with RIPA buffer (Beyotime Biotechnology). Lysate was centrifuged at 14,000g for 30 min. Supernatant fraction was used for western blot. 2ug/ml protein were subject to SDS-PAGE. Antibodies: rabbit anti-CD9 (abcam, 1:1000 dilution), rabbit anti-CD63 (abcam, 1:1000 dilution), rabbit anti-CD81 (abcam, 1:1000 dilution), rabbit anti-AKT (CST, 1:1000 dilution), rabbit anti-P-AKT (CST, 1:1000 dilution), and rabbit anti-PTEN (CST, 1:1000 dilution). Rabbit anti-GAPDH (CST, 1:1000 dilution) used as an internal control.
4
Exosomal Protein Profiling by Western Blot
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Exosomes, cells, or tissues after indicated treatments were harvested and subjected to radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China) supplemented with protease inhibitor cocktail (Roche). Purified protein was separated in 12% SDS-PAGE (120 V for stacking gel and 160 V for separation gel) and then transferred to a nitrocellulose membrane with an ice bath. The nitrocellulose membrane was blocked with 5% bovine serum albumin for 1 h and then incubated overnight with primary antibodies at 4°C. Antibodies used were mouse anti-GM130, rabbit anti-CD9, rabbit anti-TSG101, rabbit anti-PGC1α, and rabbit anti-GAPDH (all from Abcam). The membrane was then incubated for 1 h with the corresponding secondary antibodies at room temperature and visualized using the enhanced chemiluminescence (ECL) Prime western blotting detection reagent (GE Healthcare, Buckinghamshire, UK).
5
Extracellular Vesicle Protein Profiling
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The samples used for identification of widely expressed protein markers in EVs and quantification of caspase 3 and cleaved-caspase 3 expression in three groups (Blank, Dox, EV) were suspended in 100 μl radio-immunoprecipitation assay (RIPA) buffer (Solarbio, Shanghai, China), the protein of which (30 μg) was subjected to 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany). After being immersed in 5% nonfat milk for 2 h, they were incubated with primary antibodies overnight at 4 °C and secondary antibodies for 2 h at room temperature. The signal was detected by the Pierce enhanced chemiluminescence western blotting substrate (Millipore). The primary antibodies used for western blotting analysis were as follows: rabbit anti-Alix (Wanleibio, Shengyang, China), rabbit anti-CD9 (Abcam, Cambridge, UK), rabbit anti-CD63 (Wanleibio, Shengyang, China), rabbit anti-TSG101 (Abcam, Cambridge, UK), rabbit anti-caspase 3 (Wanleibio, Shengyang, China), rabbit anti-cleaved-caspase 3 (Wanleibio, Shengyang, China), and mouse anti-tubulin (Abcam, Cambridge, UK). Relevant experimental operations were following the manufacturer’s instructions.
6
Protein Extraction and Western Blot Analysis
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Proteins were extracted using RIPA lysis buffer (cat. no. HY-K1001; MedChemExpress) from JEG-3 cells and MSCs-Ex. The concentration of the isolated protein was quantified via BCA Protein Assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). Isolated proteins (5 µg) were mixed with 5X SDS-PAGE protein loading buffer (cat. no. 20315ES05; Shanghai Yeasen Biotechnology Co., Ltd.). Proteins (20 µg) were separated on 12% SDS-acrylamide gels, followed by transferring onto PVDF membranes, which were incubated with 5% non-fat milk for 1 h at room temperature, and with rabbit anti-HIF-1α (1:1,000; cat. no. ab179483; Abcam), rabbit anti-CD9 (1:1,000; cat. no. ab236630; Abcam), rabbit anti-CD81 (1:1,000; cat. no. ab79559; Abcam), rabbit anti-LAMP-2B (1:1,000; cat. no. ab18529; Abcam), rabbit anti-TSG101 (1:1,000; cat. no. ab125011; Abcam) and rabbit anti-GAPDH (1:10,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight at 4°C. Subsequently, membranes were incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG (1:5,000; cat. no. BM2006; Boster Biological Technology) for 1 h at room temperature. Protein bands were determined via ECL western blot detection reagents (Thermo Fisher Scientific, Inc.). The protein gray value was calculated using ImageJ (Version 1.5.3; National Institutes of Health).
7
Western Blot Analysis of Extracellular Vesicles
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Protein samples at a concentration of 10–30 μg were separated in 8% or 4%–15% gradient SDS-PAGE gels under reducing conditions and electroblotted onto 0.2-mm nitrocellulose membranes (GE Healthcare Life Sciences, Marlborough, MA, USA). The membranes were blocked in Tris-buffered saline-Tween 20 (TBS-T; 25 mM Tris [pH 8.0], 150 mM NaCl, and 0.05% Tween-20) containing 5% (w/v) non-fat dried milk for 1 h. After blocking, membranes were probed overnight with mouse anti-CD63, mouse anti-Alix (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-CD9 (Abcam), and mouse anti-CD81 (Becton Dickinson). After extensive washings with TBS-T, the blots were incubated with the appropriate peroxidase-conjugated secondary antibodies for 1 h at room temperature. Following incubation, the membranes were washed extensively with TBS-T, probed with enhanced SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), and detected with the ChemiDoc system (Bio-Rad, Hercules, CA, USA).
8
Exosomal Protein Profiling by Western Blotting
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Exosomal protein extracted from serum was electrophoresed and electroblotted following a standard blotting protocol. Specific primary antibodies were used, and their dilutions were as follows: mouse anti‐CD63 (Abcam, Cambridge, MA, 1:500), rabbit antiflotillin 1 (Abcam, 1:1000), rabbit anti‐CD9 (Abcam, 1:2000). HRP‐conjugated goat anti‐mouse IgG and goat anti‐rabbit IgG (Proteintech Group, Chicago, USA, both diluted 1:3000) were used as the secondary antibodies.
9
Comprehensive Protein Expression Analysis
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Cells were harvested in RIPA lysis buffer (Solarbio, Shanghai, China), quantified by a BCA Protein Assay Kit (Promega), separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). After blocking with 5% skim milk for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight. After three times washing with TBST, the membranes were incubated with secondary antibodies for 2 h at room temperature. The Pierce enhanced chemiluminescence western blotting substrate (Millipore) was used to detect the signal. The primary antibodies were used for western blot analysis: rabbit anti-Sox2 (Santa Cruz Biotechnology), rabbit anti-Oct4 (Santa Cruz Biotechnology), rabbit anti-Nanog (Bethyl), rabbit anti-P53 (Santa Cruz Biotechnology), rabbit anti-P16 (Wanleibio, Shenyang, China), rabbit anti-CD9 (Abcam, Cambridge, UK), rabbit anti-CD63 (Wanleibio, Shenyang, China), rabbit anti-IGF1R (Novus biological), mouse ant-IGF1 (Novus biological).
10
Exosome Protein Profiling by Western Blot
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Proteins were extracted from exosome and cell lysates, using radio immunoprecipitation (RIPA) buffer containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Merck Millipore, Burlington, Massahusetts, USA). The membranes were blocked with 5% non-fat milk in Tris-buffer saline and 0.1% Tween-20 and probed with the following primary antibodies: mouse anti-CD63 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-CD9 (1:2000, Abcam), mouse anti-CD81 (1:250, Invitrogen), and mouse anti-Bip/Grp78 (1:1000, BD Biosciences, San Jose, CA, USA), rabbit anti-ENG (1:1000, Abcam), rabbit anti-SEC14L2 (1:1000, Abcam), mouse anti-ZO-1 (1:1000, Thermo Fisher Scientific), mouse anti-N-cadherin (1:2000, BD bioscience), rabbit anti-Vimentin (1:5000, GeneTex, Alton, CA, USA), rabbit anti-Slug (1:1000, Cell Signaling Technology, Danvers, MA, USA), and mouse anti-GAPDH (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Chemiluminescence signals were detected using Clarity™ Western ECL Substrate and ChemiDoc (both from Bio-Rad Laboratories, Hercules, CA, USA).
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