The following antibodies (Abs) were used in the experiments of this study: anti‐CD9 (BioLegend, Cat# 312102; San Diego, CA, USA), anti‐CD63 (Novus Biologicals, Cat# NBP2‐42225; Centennial, CO, USA), anti‐CD81 (BioLegend, Cat# 349502), anti‐IGF1R (ganitumab, a kind gift from Dr. J.C. Williams), anti‐cytochrome C (BD Biosciences, Cat# 556432; Franklin Lakes, NJ, USA), anti‐syntenin (Abcam, Cat# ab19903; Waltham, MA, USA) and anti‐ApoA1 (Abcam, Cat# ab52945) Abs, as well as goat anti‐rabbit (anti‐rabbit IgG; Invitrogen, Cat# A16112; Waltham, MA, USA), IRDye 800CW goat anti‐mouse (Li‐Cor Biotechnology, Cat# 926–32210; Lincoln, NE, USA) and IRDye 680RD goat anti‐rabbit (Li‐Cor Biotechnology, Cat# 926–68071) secondary Abs. For ExoView experiments, fluorescently labelled anti‐CD9‐CF488A, anti‐CD63‐CF647 and anti‐CD81‐CF555 Abs from the manufacturer's kit were used (details below). Human lactadherin (MFG‐E8), aa Leu24‐Cys387 (contains both C1 and C2 domains) was purchased from Novus Biologicals (Cat# 2767‐MF‐050).
Anti syntenin
Manufactured by Abcam
Sourced in United States
Anti-syntenin is an antibody product used in laboratory research. It is designed to detect and bind to the syntenin protein, which plays a role in various cellular processes. The core function of this antibody is to provide a tool for researchers to study the expression and localization of syntenin in different cell types or experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd81, by Santa Cruz Biotechnology (2 mentions)
Anti calnexin, by Abcam (2 mentions)
Anti tsg101, by Abcam (2 mentions)
Anti cytochrome c, by BD (1 mentions)
Irdye 800cw goat anti mouse, by LI COR (1 mentions)
Irdye 680rd goat anti rabbit, by LI COR (1 mentions)
Anti cd9, by BioLegend (1 mentions)
Anti cd63, by Novus Biologicals (1 mentions)
Anti cd81, by BioLegend (1 mentions)
Anti apoa1, by Abcam (1 mentions)
Bsa pbs t, by Cell Signaling Technology (1 mentions)
Anti grp94, by Cell Signaling Technology (1 mentions)
Anti cd63, by Abcam (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Ez ecl kit, by Geneflow (1 mentions)
Anti alix, by Cell Signaling Technology (1 mentions)
Anti cd9, by Abcam (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Anti mitofilin, by Thermo Fisher Scientific (1 mentions)
Ab26300, by Abcam (1 mentions)
6 protocols using anti syntenin
1
Antibody Panel for Extracellular Vesicle Analysis
2
Immunoblotting for Extracellular Vesicle Markers
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Immunoblotting was performed as we previously described [9 (link)], loading 10 µg of protein per lane of the gel. Primary antibodies which are anti-CD63 (1/500; Abcam, Cat.#:ab68418), anti-syntenin (1/1000; Abcam, Cat.#:ab133267), anti-TSG101 (1/1000; Abcam, Cat.#:ab83), anti-CD81 (1/250; Santa Cruz, Cat.#: 5A6), anti-GRP94 (1/2000; Cell Signalling, Cat.#:2104 S), and anti-calnexin (1/1000; Abcam, Cat.#:ab92573) were used. Secondary antibodies used were anti-mouse (1/1000 in 5% BSA/PBS-T, Cell Signalling, Cat.#:7076) or anti-rabbit (1/1000 in 5% BSA/PBS-T, Cell Signalling, Cat.#:7074). Lysate of Hs578Ts(i)8 cells were included on all gels as a control and densitometric analysis was performed using Fiji software. Of note: cropped images of blots are included in the main manuscript and the associated full blots can be found in the Supplemental Material.
3
Exosome Protein Marker Detection
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The samples were tested for the presence of exosome protein markers using immunoblotting. Briefly, 10 μg of protein were loaded onto a NuPAGE Novex 4-12% Bis-Tris Gel (ThermoFisher Scientific/Invitrogen) then run in NuPAGE MOPS SDS Running Buffer (ThermoFisher Scientific/Invitrogen) at 150 V for 1 hour. The proteins were electroblotted for 1 hour at 25 V onto a methanol-activated polyvinylidene difluoride membrane (Bio-Rad Laboratories). The protein transfer was ascertained by Ponceau Red stain (Merck). Antibodies used for immunostaining were anti-CD9 (Abcam), anti-Alix (Cell Signaling Technology), and anti-syntenin (Abcam). The syntenin band was visualized using the EZ-ECL kit (Geneflow), and the Alix and CD9 bands were visualized using the Westar Supernova ECL kit (Cynagen) with the Gel-Box imaging system (Sygene), or on Kodak films according to the manufacturer’s instructions.
4
Western Blot Analysis of MSC-Derived Extracellular Vesicles
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For Western blot analysis, the pre-conditioned MSCs and their corresponding separated mEVs and sEVs were resuspended in RIPA buffer (1% NONIDET p-40, 0.1% SDS, 0.1% Sodium deoxycholate, protease inhibitor cocktail 1x, in PBS pH 7.5) and protein content was quantified by BCA assay. Afterwards, 10 μg of proteins for each sample was loaded on 4-20% Mini-PROTEAN®TGX™ Precast Gel for electrophoresis, and proteins were blotted onto a PVDF membrane with a semi-dry transfer system (all from Bio-Rad Europe, Basel, Switzerland). Blot membrane was incubated overnight at 4 • C with the following specific primary antibodies: anti-syntenin (1:1000 dilution, ab133267 Abcam, USA), anti-mitofilin (1:1000 dilution, PA5 89627, Invitrogen, USA) and anti-Grp94 (1:1000 dilution, ab238126, Abcam, USA), and anti-lamin A (1:1000 dilution, ab26300, Abcam, USA) prepared in odyssey blocking buffer (LI-COR, USA) at the dilution as recommended by the manufacturer. Following, membranes were incubated with appropriate secondary antibodies such as IRDye® 680RD or 800CW goat anti-mouse or goat anti-rabbit secondary Ab (LI-COR Biosciences, Lincoln, Nebraska, USA). Infrared signal was detected using Odyssey CLx Detection System (LI-COR Biosciences).
5
Characterization of Extracellular Vesicles from Human iPSCs and MSCs
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Human iPSCs, MSCs and purified EVs were lysed in RIPA Buffer (ThermoFisher Scientific) supplemented with protease and phosphatase inhibitor cocktails (Roche, Nutley, NJ), followed by centrifugation at 16,000 g for 20 minutes at 4°C. Protein quantification of cell and EV lysates was conducted using a Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Lysates were resolved using a 4%–12% NuPage gel, then transferred onto nitrocellulose membrane (GE Healthcare, California, USA). Blots were probed using primary antibodies in 1× TBST (Tris‐buffered Saline containing 0.1% Tween‐20% and 5% Blotting grade blocker) purchased from Bio‐Rad. Primary antibodies used included anti‐CD81 (Santa Cruz, Cat #SC166029, Dallas, TX), anti‐Calnexin (Abcam, Cat #ab22595, Cambridge, MA), anti‐TSG101 (Abcam, Cat #ab125011), anti‐Syntenin (Abcam, Cat #ab133267), anti‐PRDX1 (ThermoFisher Scientific, Cat #LF‐MA0073), anti‐PRDX2 (ThermoFisher Scientific, Cat #LF‐MA0144), and anti‐GAPDH (Cell Signaling Technology, Cat #5174). Secondary antibodies used included goat anti‐mouse IgG (H + L; LI‐COR Biosciences, Cat #P/N 925–32,210, Lincoln, NE) and goat anti‐rabbit IgG (H + L; LI‐COR Biosciences, Cat #P/N 925–32,211).
6
Western Blot Analysis of Extracellular Vesicles
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Briefly, 35 µL of EVs isolated from mice plasma were denatured directly in 5× loading buffer and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% Tris-glycine gel. The separated proteins were transferred onto nitrocellulose membrane, and after blocking with 5% non-fat milk powder (w/v) in Tris-buffered saline with tween (TBS-T, 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20), the fresh membrane or stripped membrane was probed with anti-syntenin (Abcam, Waltham, MA) or anti-CD63 (Invitrogen, Waltham, MA) or anti-GOLGA2 (Novus Biologicals, Littleton, CO) primary antibodies for overnight incubation at 4 ℃. Further, in each case, the membrane was washed 3 times with TBS-T and incubated with appropriate secondary antibodies before visualization by the ECL detection system. The autoradiograms/ bands were scanned and quantified using Image J (version 1.53e) using corresponding bands in Ponceau stained membrane as the loading control.
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