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) (Catalog ID 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

Protocol for Detection of Proteins inside Exosome

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.

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.