Extracellular Vesicle Protein Profiling

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.

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Malkin E.Z., De Michino S., Lambie M., Gill R., Zhao Z., Rostami A., Arruda A., Minden M.D, & Bratman S.V. (2022). Cell-free DNA topology depends on its subcellular and cellular origins in cancer. JCI Insight, 7(20), e159590.

Publication 2022

AnykD Mini-PROTEAN TGX Pre-Cast Gel Trans-Blot Turbo ab133497 ab19903 ab1791 Odyssey CLx

A protein Anti antibody Antibodies Antibody Assay Cast Conditioned media Extracellular vesicles Flotillin1 Goat Histone h3 Hsp90 Irdye 800cw Membranes Milk Mouse Protein Pvdf Rabbit Saline Tween 20 Western blot

Corresponding Organization : University Health Network

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Variable analysis

independent variables

Concentrated conditioned media samples

dependent variables

Relevant protein markers

control variables

Bradford assay to determine protein concentrations
5 μg of each sample run on an AnykD Mini-PROTEAN TGX Pre-Cast Gel at 135 V for 50 minutes
Protein transferred to a PVDF membrane using a Bio-Rad Trans-Blot Turbo
Membranes blocked in 5% skim milk for 1 hour at room temperature
Incubation with primary antibodies (anti-HSP90, anti-CD63, anti-flotillin1, anti-syntenin1, and anti–histone H3) overnight at 4°C
Incubation with corresponding IRDye 800CW goat anti-mouse or anti-rabbit secondary antibody for 1 hour at room temperature

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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