EV-Perm(TM) Permeabilization Pretreatment Kit for Exosome Membrane

EV-Perm™ Permeabilization Pretreatment Kit for Exosome Membrane 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 exosome 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

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) 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 Solution to 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 luminal proteins 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.