Cell lysates were prepared in M-PER buffer (Thermo Fisher Scientific, 78501) supplemented with protease inhibitors (Roche). For Western blotting, cell lysates and EVs were resuspended in NuPAGE LDS sample buffer (Novex, NP0008) with NuPAGE sample reducing agent (Novex, NP0009). IP was carried out using the FLAG immunoprecipitation kit (Sigma-Aldrich). Cell lysates were incubated in anti-FLAG agarose affinity gel at 4°C overnight. Immunoprecipitated samples were washed three times with washing buffer, eluted, and subjected to Western blotting. All Western blotting samples were loaded on a 4 to 12% NuPAGE gel or 12% NuPAGE gel and transferred onto 0.22-μm polyvinylidene difluoride membrane (Bio-Rad, 1620177). Primary antibodies include anti-FLAG antibody (Sigma-Aldrich, F1804; 1:2000 dilution), anti-vinculin antibody (Abcam, ab129002; 1:2000 dilution), anti-CD9 antibody (Cell Signaling Technology, 1317S; 1:1000 dilution), anti-Scamp3 antibody (GeneTex, GTX102216; 1:2000 dilution), and anti-ARRDC1 antibody (in-house; 1:3000 dilution). Secondary antibodies include horseradish peroxidase (HRP)–conjugated anti-rabbit (Cell Signaling Technology, 7074S; 1:2000 dilution) and HRP-conjugated anti-mouse (Cell Signaling Technology, 7076S; 1:2000 dilution).
Anti cd9 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-CD9 antibody is a research-use only product designed to detect the CD9 protein. CD9 is a member of the tetraspanin family of proteins, which play roles in various cellular processes. The antibody can be used to identify and study CD9-expressing cells and its expression patterns.
Automatically generated - may contain errors
Lab products found in correlation
Exoquick, by System Biosciences (2 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti vinculin antibody, by Abcam (1 mentions)
M per buffer, by Thermo Fisher Scientific (1 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
0.22 μm polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Flag immunoprecipitation kit, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
Anti alix antibody, by Cell Signaling Technology (1 mentions)
Anti caveolin 1 antibody, by Cell Signaling Technology (1 mentions)
Zetaview software, by Particle Metrix (1 mentions)
Tsg101, by Abcam (1 mentions)
3 protocols using anti cd9 antibody
1
Immunoprecipitation and Western Blotting
2
EV Cargo Protein Extraction and Analysis
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Cargo proteins were extracted from EVs by heating the vesicles at 60 °C for 15 min in 100 mM TrisHCl buffer containing 4% SDS. Proteins released from the ruptured vesicles were then mixed with gel loading buffer and boiled at 95 °C for 10 min. Protein samples were separated by SDS-PAGE on 12% polyacrylamide gels and then transferred onto nitrocellulose membranes at 100 V for 1 h. The membranes were probed with primary antibodies at 4 °C overnight. The antibodies used for Western blots were: anti-Alix antibody (#2171), anti-flotillin-1 antibody (#18634), anti-CD9 antibody (#13174), anti-caveolin-1 antibody (#3267S) from Cell Signaling Technologies (Danvers, MA, USA). The anti-hepatocyte growth factor receptor antibody (ab59884) and anti-FTH1 (ab75973) antibodies were from Abcam (Cambridge, UK). Anti-E-cadherin (sc-21791), anti-N-cadherin (sc-59987), anti-cathepsin B (sc-365558), anti-cathepsin D (sc-377299) and anti-IGFBP3 (sc-9028) antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-PLOD2 (408105) antibody was obtained from Thermo Fisher Scientific. Protein–antibody conjugates were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific).
3
Exosome Isolation and Characterization
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ExoQuick (ExoQuick, SBI, USA) was used for the extraction of exosomes according to the manufacturer's protocol. ExoQuick (63 μL) was added to 250 μL of serum sample and incubated for 30 minutes at 4°C to precipitate exosomes. Subsequently, the mixture was centrifuged at 1500 g for five minutes at 4°C, followed by another centrifugation at 1500 g for five minutes at 4°C, to eliminate contaminating apoptotic bodies and cellular debris. The precipitates were collected, resuspended in 50 μL of phosphate‐buffered saline (PBS), and stored at −80°C until further use.
Transmission electron microscopy (TEM) was used to analyze the concentration and size distribution of exosomes using nanoparticle tracking analysis (NTA). Exosomal suspensions in PBS were analyzed using ZetaView software (Particle Metrix, Germany) according to the manufacturer's instructions. Filtered PBS was used as the control. Total proteins were extracted from serum exosomes and exosome‐depleted supernatants (EDS); western blotting was performed according to the manufacturer's instructions. Antibodies against exosome‐specific proteins including anti‐CD9 antibody (#13403, Cell Signaling Technology, ) and TSG101 (#ab125011, Abcam) were used to detect the exosome‐specific proteins.
Transmission electron microscopy (TEM) was used to analyze the concentration and size distribution of exosomes using nanoparticle tracking analysis (NTA). Exosomal suspensions in PBS were analyzed using ZetaView software (Particle Metrix, Germany) according to the manufacturer's instructions. Filtered PBS was used as the control. Total proteins were extracted from serum exosomes and exosome‐depleted supernatants (EDS); western blotting was performed according to the manufacturer's instructions. Antibodies against exosome‐specific proteins including anti‐CD9 antibody (#13403, Cell Signaling Technology, ) and TSG101 (#ab125011, Abcam) were used to detect the exosome‐specific proteins.
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