EV-Perm™ Permeabilization Pretreatment Kit for Exosome Membrane
EV-Perm™ Permeabilization Pretreatment Kit for Exosome Membrane can detect proteins inside extracellular vesicles (EVs), including exosomes. Until now, protein detection using the PS Capture™ Exosome ELISA Kit series and PS Capture™ Exosome Flow Cytometry Kit has been limited to surface markers of exosomes. This product enhances the permeability of the exosome membrane, thus allowing antibodies to penetrate and detect marker proteins within the exosome lumen.
Product Overview
This Kit enhances the membrane permeability of extracellular vesicles (EVs), including exosomes. It enables the detection of proteins in the exosome lumen using antibodies.
[Note]
*This kit is intended to be used in conjunction with the PS Capture™ Exosome ELISA Kit or PS Capture™ Exosome Flow Cytometry Kit. Detection antibodies must be obtained separately.
Features
- Enhances the membrane permeability of exosomes using the three reagents included in the kit.
- Enables the detection of proteins in the exosome lumen when used in conjunction with an ELISA kit or a flow cytometry kit.
- Compatible not only with purified exosomes, but also with cell culture supernatants and body fluid samples.
Samples
- Purified exosomes (extracellular vesicles)
- Cell culture supernatants
- Body fluid samples (serum and plasma)
Principle
Protocol
Detection of Proteins inside Exosome
Below is the protocol for detection of proteins inside exosomes using the EV-Perm™ Exosome Membrane Penetration Kit in conjunction with the PS Capture™ Exosome ELISA Kit (Streptavidin HRP) (Product Number: 298-80601). Please refer to the product insert on the product details page for more a detailed description of procedures.
(Youtube, 05:22)
1. Capture of Exosomes
1. Wash each well of the Exosome Capture 96 Well Plate with washing buffer (1x).
2. Dispense the diluted samples into each well.
3. Incubate for at least 2 hours using a microplate shaker.
4. Wash with washing buffer (1x).
2. Enhancement of Membrane Permeability
1. Add the permeabilization reagent or negative control reagent to each well.
2. Incubate for 1 hour using a microplate shaker.
3. Wash with washing buffer (1x).
3. Detection of Target Protein
3-1. Primary Antibody Reaction
1. Add the biotinylated antibody against the target protein to each well.
2. Incubate for 1 hour using a microplate shaker.
3. Wash with washing buffer (1x).
3-2. HRP-Streptavidin Reaction
1. Add HRP-conjugated streptavidin (1x)(1×) to each well.
2. Incubate for 2 hours using a microplate shaker.
3. Wash with washing buffer (1x).
3-3. Color Development Reaction
1. Add TMB Solutionto each well and shake for about 1 minute using a microplate shaker.
2. Allow to stand for 30 minutes.
3. Add Stop Solution (pre-equilibrated to room temperature).
4. Immediately measure absorbance at 450 nm and at a secondary wavelength of 620 nm (600-650 nm).
Components of PS Capture™ Exosome ELISA Kit (Streptavidin HRP)
Components of EV-Perm™ Exosome Membrane Penetration Kit
Application Data
Detection of proteins inside exosomes by ELISA
Exosomes purified from HEK293T and H4 cell culture supernatants were permeabilized with the reagents in this kit, and the proteins inside exosomes Alix and α-synuclein, respectively, were detected using the PS Capture™ Exosome ELISA Kit (Streptavidin HRP).
[Result] With permeabilization, Alix and α-synuclein inside exosomes were detected by ELISA.
Detection of proteins inside exosomes by Flow Cytometry
Exosomes purified from HEK293T cell culture supernatant were permeabilized with the reagents in this kit, and Alix that is the protein inside EVs, and CD63 that is exosome surface marker protein were detected using the PS Capture™ Exosome Flow Cytometry Kit.
[Result] With permeabilization, Alix inside exosomes was detected by Flow Cytometry.
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For research use or further manufacturing use only. Not for use in diagnostic procedures.
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