The following primary antibodies were used: goat polyclonal anti-adiponectin (AF1119, R&D Systems); goat polyclonal anti-T-cadherin (AF3264, R&D Systems); rabbit monoclonal anti-α-tubulin (11H10, Cell Signaling Technology); sheep polyclonal anti-human MFG-E8 (AF2767, R&D Systems); mouse monoclonal anti-human CD63 (H5C6, BD Biosciences); rabbit monoclonal anti-Tsg101 (ab125011, R&D Systems); rabbit polyclonal anti-syntenin (ab19903, Abcam); and mouse monoclonal anti-ALIX (3A9, Santa Cruz Biotechnology). The following secondary antibodies were used: horseradish-peroxidase-conjugated (HRP-conjugated) rabbit anti-sheep immunoglobulin G (IgG) (Invitrogen); HRP-conjugated donkey anti-goat IgG (R&D systems); and HRP-conjugated sheep anti-mouse IgG antibodies and donkey anti-rabbit IgG antibody (GE Healthcare).
Ab19903
Manufactured by Abcam
Sourced in United States
Ab19903 is a primary antibody product manufactured by Abcam. It is designed for laboratory research use.
Automatically generated - may contain errors
Lab products found in correlation
Af1119, by R&D Systems (2 mentions)
Af3264, by R&D Systems (2 mentions)
Ab30871, by Abcam (2 mentions)
Ab77581, by Santa Cruz Biotechnology (2 mentions)
Ab217345, by Abcam (2 mentions)
A16110, by Thermo Fisher Scientific (2 mentions)
Bistris bolt mini gel, by Thermo Fisher Scientific (2 mentions)
Epr15314 ab186429, by Abcam (2 mentions)
Epr21151, by Abcam (2 mentions)
A18745, by Thermo Fisher Scientific (2 mentions)
Pierce elc western substrate, by Thermo Fisher Scientific (2 mentions)
Ai600 imager, by GE Healthcare (2 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Ab133497, by Abcam (2 mentions)
Ab117600, by Abcam (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Ab92726, by Abcam (2 mentions)
Chemidoc touch, by Bio-Rad (2 mentions)
Gradient sds page gel, by Bio-Rad (2 mentions)
14 protocols using ab19903
1
Antibody-Based Protein Detection Protocols
2
Western Blot Analysis of Extracellular Vesicles
3
Extracellular Vesicle Protein Profiling
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Concentrated conditioned media samples were subjected to Western blot to visualize relevant protein markers. First, IP was performed on samples as described above. A Bradford assay was conducted to determine protein concentrations, and 5 μg of each sample was run on an AnykD Mini-PROTEAN TGX Pre-Cast Gel (Bio-Rad) at 135 V for 50 minutes. Protein was transferred to a PVDF membrane using a Bio-Rad Trans-Blot Turbo. Membranes were blocked in 5% skim milk for 1 hour at room temperature and incubated with the following primary antibodies overnight at 4°C: anti-HSP90 (37-9400; mouse monoclonal; Thermo Fisher Scientific), anti-CD63 (25682-1-AP; rabbit polyclonal; Thermo Fisher Scientific), anti-flotillin1 (ab133497; rabbit monoclonal; Abcam), anti-syntenin1 (ab19903; rabbit polyclonal; Abcam), and anti–histone H3 (ab1791; rabbit polyclonal; Abcam); these markers were selected based on the Minimal Information for Studies of Extracellular Vesicles 2018 guidelines (19 (link)) and a recent comprehensive overview of EV protein markers (53 (link)). Following primary antibody incubation, membranes were washed in 1× Tris-buffered saline with Tween 20 (TBS-T) and incubated with the corresponding IRDye 800CW goat anti-mouse or anti-rabbit secondary antibody (LI-COR Biosciences) for 1 hour at room temperature. Membranes were subsequently washed in 1× TBS-T and imaged on a LI-COR Odyssey CLx.
4
Modulation of Extracellular Vesicle Composition
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Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200. Syntenin-1 (rabbit; 610821; BD) and flotillin-1 (rabbit; ab19903; Abcam) mouse antibodies were used at 1:250. Antibodies against SNAP23 (rabbit; 111-202; Synaptic Systems), GFP (rabbit; A01388; Genscript), and β-actin (mouse; sc-47778; Santa Cruz Biotechnology, Inc.) were used at 1:1,000. Secondary anti–mouse Alexa Fluor 594 antibody (goat; A-11032; Invitrogen) was used at 1:500. For Western blotting, cells or exosomes were lysed in a 1% SDS buffer, and equal amounts of protein were loaded onto an SDS-PAGE gel. Only gels for CD63, CD81, and CD9 detection were run under nonreducing conditions.
EGTA-AM (Sigma-Aldrich) was applied at 200 µM for 15 min at 37°C, BAPTA-AM (Sigma-Aldrich) was applied at 20 µM for 15 min at 37°C, and GÖ6983 (Axon Medchem) and GÖ6976 (EMD Millipore) were applied at 1 µM for 2 h at 37°C. GW4869 (Sigma-Aldrich) was applied at 5 µM for 4 h. Histamine (Sigma-Aldrich) was used at 100 µM, and caffeine (Sigma-Aldrich) was used at 20 mM. UBO-QIC (a gift from E. Kostenis, University of Bonn, Bonn, Germany) was used at 1 µM for at least 30 min (preincubation).
EGTA-AM (Sigma-Aldrich) was applied at 200 µM for 15 min at 37°C, BAPTA-AM (Sigma-Aldrich) was applied at 20 µM for 15 min at 37°C, and GÖ6983 (Axon Medchem) and GÖ6976 (EMD Millipore) were applied at 1 µM for 2 h at 37°C. GW4869 (Sigma-Aldrich) was applied at 5 µM for 4 h. Histamine (Sigma-Aldrich) was used at 100 µM, and caffeine (Sigma-Aldrich) was used at 20 mM. UBO-QIC (a gift from E. Kostenis, University of Bonn, Bonn, Germany) was used at 1 µM for at least 30 min (preincubation).
5
Protein Extraction and Western Blot Analysis
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Samples (lung tissue or lung vascular
endothelial cells) were dissolved in lysis buffer for lysis, centrifuged
at 12,000g for 5 min to collect their supernatants,
and analyzed for their protein concentration using a BCA kit. High-resolution
separation of protein samples (20 or 40 μg protein/well) was
obtained on a 12% SDS-PAGE gel. These proteins were then transferred
to nitrocellulose or PVDF membranes (Milipore). These membranes were
blocked for 10 min at room temperature using a commercial blocking
solution (PS108, Epizyme Biotech, Shanghai, China), washed three times
with TBST, and incubated with VE-cadherin (1:1000, ab205336, Abcam),
ZO-1 (1:1000, ab216880, Abcam), ICAM-1 (1:1000, ab222736, Abcam),
β-catenin (1:20000, Affinity), ITGAM (1:1000, #17800, CST),
ITGB2 (1:1000, #72607, CST), TSG101 (1:1000, ab125011, Abcam), CD9
(1:1000, A1703, Abclonal), Calnexin (1:1000, ab227310, Abcam), and
Syntenin (1:1000, ab19903, Abcam), overnight at 4 °C (approximately
12 h). These membranes were then washed three times with TBST and
incubated with a fluorescently labeled secondary antibody (antirabbit
or antimouse lgG, Cell Signaling Technology) for 60 min at room temperature.
After three washes, the prints were visualized using an imaging system
(Bio-Rad).
endothelial cells) were dissolved in lysis buffer for lysis, centrifuged
at 12,000g for 5 min to collect their supernatants,
and analyzed for their protein concentration using a BCA kit. High-resolution
separation of protein samples (20 or 40 μg protein/well) was
obtained on a 12% SDS-PAGE gel. These proteins were then transferred
to nitrocellulose or PVDF membranes (Milipore). These membranes were
blocked for 10 min at room temperature using a commercial blocking
solution (PS108, Epizyme Biotech, Shanghai, China), washed three times
with TBST, and incubated with VE-cadherin (1:1000, ab205336, Abcam),
ZO-1 (1:1000, ab216880, Abcam), ICAM-1 (1:1000, ab222736, Abcam),
β-catenin (1:20000, Affinity), ITGAM (1:1000, #17800, CST),
ITGB2 (1:1000, #72607, CST), TSG101 (1:1000, ab125011, Abcam), CD9
(1:1000, A1703, Abclonal), Calnexin (1:1000, ab227310, Abcam), and
Syntenin (1:1000, ab19903, Abcam), overnight at 4 °C (approximately
12 h). These membranes were then washed three times with TBST and
incubated with a fluorescently labeled secondary antibody (antirabbit
or antimouse lgG, Cell Signaling Technology) for 60 min at room temperature.
After three washes, the prints were visualized using an imaging system
(Bio-Rad).
6
Profiling EV Protein Markers
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MSC-EVs lysates were prepared using 30X ethanol precipitation into RIPA lysis buffer. Lysates were volume loaded and separated on a 12% Bolt Bis-Tris Gel and probed using Abcam antibodies against flotillin-1 (ab133497 at 1:10,000 dilution), Annexin-2 (ab41803 at 1:1000 dilution), Syntenin-1 (ab19903 at 1:1000 dilution), MHC-I (ab110645 at 1:1000 dilution), MHC-II (157210 at 1:10000 dilution), and Calreticulin (ab92516 at 1:1000 dilution).
7
Quantification of Extracellular Vesicle Proteins
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Neurons on 10-cm plates were washed 3 times in PBS and lysed in lysis buffer (20 mM Tris pH 7.4, 100 mM NaCl, 5 mM EDTA, 1% Triton-X 100, supplemented with 1 × HALT protease and phosphatase inhibitor cocktail (ThermoScientific, 78,440). Following centrifugation of EVs from neuron-conditioned media, pellets were resuspended in 25 µL lysis buffer. SDS-PAGE sample loading buffer was added to samples which were subsequently resolved by SDS-PAGE and processed for Western blotting. Primary antibodies used include rabbit polyclonal anti-syntenin (Abcam, ab19903, RRID:AB_445200, 1:1,000), mouse monoclonal anti-flotillin-1 (BD Biosciences, 610,821, RRID:AB_398140, 1:1,000), mouse monoclonal anti-gp96 (R&D systems, MAB7606, clone 816,803, 1:500), and mouse monoclonal anti-β3-tubulin (Biolegend, 801,201, RRID:AB_2313773, 1:5,000). HRP-conjugated affinity purified secondary anti-rabbit and anti-mouse were purchased from Jackson Immunoresearch Labs.
8
Western Blot Analysis of Protein Expression
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Twenty micrograms of total protein were resolved on 4–20% Tris HCl polyacrylamide gels using a Criterion electrophoresis system (BioRad; Hercules, CA, USA) at 200 V for 50 min at room temperature. The separated proteins were subjected to transfer onto nitrocellulose membranes (Thermo Fisher Scientific) by using a Criterion transfer system (Bio-Rad). The membranes were blocked in 5% nonfat dry milk 1× Tris-buffered saline (TBS) (Bio-Rad) (w/v) for 1 h at room temperature before being incubated in a 1:1000 dilution of primary antibody (anti-HSP70 (ab181606; Abcam, Waltham, MA, USA, annexin A2 (8235; Cell Signaling Tech, Danvers, MA, USA), GAPDH (649203; Biolegend, San Diego, CA, USA), syntenin (ab19903; Abcam) while rocking overnight at 4 °C. Thereafter, the membranes were washed three times with 1× TBS for 5 min intervals and then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody at a dilution of 1:3000 prepared in blocking solution while on a rocker for 1 h at room temperature. The membranes were then washed four times with 1× TBS for 4 min intervals, incubated with ECL reagent (BioRad) for 7 min at room temperature, and then developed (Bio-Rad imager).
9
Exosomal Protein Profiling by Western Blot
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Exosomes or whole cell lysates were loaded onto 4–20% gradient SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes. The membranes were blocked with Block-OneTM blocking reagent (Nacalai Tesque) and then incubated with primary antibodies using Can Get SignalTM solution 1 (TOYOBO) overnight at 4 °C and followed by incubation with secondary antibodies conjugated with HRP using Can Get SignalTM solution 2 (TOYOBO) for 60 minutes at room temperature. The following primary antibodies were used: goat polyclonal anti-adiponectin (AF1119, R&D); goat polyclonal anti-T-cadherin (AF3264, R&D); rabbit monoclonal anti-α-tubulin (11H10, Cell Signaling); rat monoclonal anti-mouse CD63 (clone R5G2, MBL); rabbit polyclonal anti-syntenin (ab19903, abcam); rabbit polyclonal anti-Tsg101 (ab125611, abcam); goat polyclonal anti-MFG-E8 (AF2805, R&D); mouse monoclonal anti-Alix (ab117600, abcam); rabbit polyclonal anti-Nox2/gp91 phox (ab80508, abcam). CD63 was detected under non-reducing conditions. Chemiluminescence signals developed with Chemi-Lumi One SuperTM (Nacalai Tesque) were visualized by ChemiDoc TouchTM and quantitated using Image Lab software (Bio-Rad).
10
Exosome Isolation and Analysis from RAW 264.7 Cells
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RAW 264.7 cells were plated at 5 × 107 cells per plate in 10 cm dishes. After 1 or 2 days in culture, cell culture medium was collected, and cells were washed once with PBS and harvested by scraping. For whole-cell lysates (WCLs), cells were pelleted and lysed directly in 1× Laemmli sample buffer with fresh β-mercaptoethanol (Bio-Rad), sonicated to break up DNA, and boiled for 5 min. Culture medium was precleared of dead cells and cell debris by spinning for 5 min at 3,000 × g. Exosomes were then collected by ultracentrifugation for 1 h at 100,000 × g. Exosome pellets were resuspended directly in 1× sample buffer and boiled for 5 min. Proteins from WCLs and exosomes were resolved and imaged by SDS-PAGE and Western blot analysis as described above using antibodies for FLAG (Sigma, F-1804; 1:5,000), Alix (Abcam, ab117600; 1:2,500), and syntenin-1 (Abcam, ab19903; 1:2,500).
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