sEV protein content was measured by BCA, and 10 μg aliquots were used for Western blot analysis. sEVs were lysed in Laemmli buffer at 96°C for 5 minutes to denature proteins, and protein extracts were resolved by SDS-PAGE. Antibodies were tested against albumin (sc-271605, Santa Cruz Biotechnology), apolipoprotein B (ab139401, Abcam), TSG101 (ab125011, Abcam), CD9 (ab58989, Abcam), and ALIX (ab88743, Abcam). Antibodies were chosen according to MISEV 2023 guidelines (21 (link)). Immunostaining against PAX8 (PA 0300, Biopat), a well-known ovarian carcinoma marker, was also incorporated in the Western blot characterization panel. The biological validation used antibodies against STX5 (HPA001358, Sigma-Aldrich) and S100A4 (ab93283, Abcam). Clarity ECL Western Blotting Substrate (Bio-Rad) was used to visualize the bands and developed on the ChemiDoc imaging system (Bio-Rad). The intensity of the immunoreactive bands was quantified by densitometry using ImageJ (NIH).
Ab58989
Manufactured by Abcam
Sourced in United Kingdom
Ab58989 is a laboratory equipment product. It is a core function device used for specific laboratory applications. No further details can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab125011, by Abcam (1 mentions)
Ab139401, by Abcam (1 mentions)
Ab88743, by Abcam (1 mentions)
Ab93283, by Abcam (1 mentions)
Sc 271605, by Santa Cruz Biotechnology (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Ab52894, by Abcam (1 mentions)
Ab75813, by Abcam (1 mentions)
Ab38496, by Abcam (1 mentions)
Ab59477, by Abcam (1 mentions)
2 protocols using ab58989
1
Characterization of Extracellular Vesicles
2
Exosome Capture Microarray Protocol
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Printing of Abs to be used for exosome capture in microarrays was performed as described in Supplemental Methods . The following Abs were used for capture of total exosomes: anti-CD9 (ab58989, Abcam, Cambridge, UK), anti-CD63 (NBP2-42225AF647, Novusbio), anti-CD81 (ab59477, Abcam). The Abs cocktail for the capture of tumor-enriched exosomes (TEX) consisted of anti-EGFR1 (ab52894, Abcam), anti-MAGEA3 (ab38496, Abcam), anti-EpCAM (ab75813, Abcam) and anti-CSPG4 (a gift from Dr. Soldano Ferrone, Harvard Medical School). As positive and negative controls, human IgG and PBS were used. Figure 2(a ) is a graphic illustrating the strategy used for capture of TEX from plasma-derived total exosomes. Titrations of optimal Ab concentrations for TEX capture and testing of microarrays specificity for TEX are illustrated in Figure 2(b ) and described in Supplemental Methods.
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