Purified EVs were lysed with 1% SDS 50 mM Tris pH 7.6-lysis solution, mixed with SDS sample buffer, and loaded onto 8% acrylamide gels (10 μl). Gels were transferred to nitrocellulose membranes (0.45 μm, Biorad) and blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween 20 (TBS-T) for 1–2 h. Membranes were probed with antibodies for EV markers, anti-CD63 (1:1000, Abcam, ab59479), anti-Flotillin (1:1000, BD, 610821), and anti-Alix (1:1000 Sigma, SAB 4200476). As a negative control, anti-Calnexin (1:1000, Cell Signaling, mAb 2679) was used. Appropriate secondary antibodies were added and detected by ECL (Thermo Scientific, 32,106 and 34,095). The same procedure was applied to detect integrins and ECM proteins such as fibronectin (Abcam, ab2413) and collagen (Abcam, ab34710).
Ab59479
Manufactured by BD
Ab59479 is a laboratory equipment product. It serves a core function in research and analysis applications. Further details about its specific use or capabilities are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab2413, by Abcam (3 mentions)
Ab34710, by Abcam (3 mentions)
Sab 4200476, by Merck Group (3 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Fcf 200 cu, by Electron Microscopy Sciences (2 mentions)
Mab 2679, by Cell Signaling Technology (2 mentions)
Anti calnexin, by Cell Signaling Technology (1 mentions)
3 protocols using ab59479
1
Characterizing Extracellular Vesicle Composition
2
Characterization of Purified Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Transmission electron microscopy: For negative staining of puri ed SEVs, 5 mL of EV samples was added to Formvar carbon lm-coated grids (FCF-200-Cu; Electron Microscopy Sciences; Hat eld, PA) for 60 sec. Grids were immediately xed with 4% paraformaldehyde in water for 20 min, stained with 2% unranyl acetate for 2 min, and allowed to air-dry. For each step, the excess of solution was removed by wicking with a lter paper. The grids were imaged using a TEM Tecnai F20 G2, 200Kv in 40000 x magni cation.
Western blotting: Puri ed EVs were lysed with 1% SDS 50mM Tris pH 7.6-lysis solution, mixed with SDS sample buffer, and loaded onto 8% acrylamide gels (10 ml). Gels were transferred to nitrocellulose membranes and blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween 20 (TBS-T) for 1-2 h. Membranes were probed with antibodies for EV markers, anti-CD63 (Abcam, ab59479), anti-Flotillin (BD, 610821), and anti-Alix (Sigma, SAB 4200476). As a negative control, anticalnexin (Cell Signaling, mAb 2679) was used. Appropriate secondary antibodies were added and detected by ECL (Thermo Scienti c, 32106 and 34095). The same procedure was applied to detect integrins and ECM proteins, bronectin (Abcam, ab2413) and collagen (Abcam, ab34710).
Western blotting: Puri ed EVs were lysed with 1% SDS 50mM Tris pH 7.6-lysis solution, mixed with SDS sample buffer, and loaded onto 8% acrylamide gels (10 ml). Gels were transferred to nitrocellulose membranes and blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween 20 (TBS-T) for 1-2 h. Membranes were probed with antibodies for EV markers, anti-CD63 (Abcam, ab59479), anti-Flotillin (BD, 610821), and anti-Alix (Sigma, SAB 4200476). As a negative control, anticalnexin (Cell Signaling, mAb 2679) was used. Appropriate secondary antibodies were added and detected by ECL (Thermo Scienti c, 32106 and 34095). The same procedure was applied to detect integrins and ECM proteins, bronectin (Abcam, ab2413) and collagen (Abcam, ab34710).
3
Characterization of Extracellular Vesicle Composition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transmission electron microscopy: For negative staining of puri ed SEVs, 5 μl of EV samples was added to Formvar carbon lm-coated grids (FCF-200-Cu; Electron Microscopy Sciences; Hat eld, PA) for 60 sec. Grids were immediately xed with 4% paraformaldehyde in water for 20 min, stained with 2% uranyl acetate for 2 min, and allowed to air-dry. For each step, the excess of solution was removed by wicking with a lter paper. The grids were imaged using a TEM Tecnai F20 G2, 200Kv in 40000 x magni cation.
Western blotting: Puri ed EVs were lysed with 1% SDS 50mM Tris pH 7.6-lysis solution, mixed with SDS sample buffer, and loaded onto 8% acrylamide gels (10 ml). Gels were transferred to nitrocellulose membranes (0.45 mm, Biorad) and blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween 20 (TBS-T) for 1-2 h. Membranes were probed with antibodies for EV markers, anti-CD63 (1:1000, Abcam, ab59479), anti-Flotillin (1:1000, BD, 610821), and anti-Alix (1:1000 Sigma, SAB 4200476). As a negative control, anti-calnexin (1:1000, Cell Signaling, mAb 2679) was used. Appropriate secondary antibodies were added and detected by ECL (Thermo Scienti c, 32106 and 34095). The same procedure was applied to detect integrins and ECM proteins such as bronectin (Abcam, ab2413) and collagen (Abcam, ab34710).
Western blotting: Puri ed EVs were lysed with 1% SDS 50mM Tris pH 7.6-lysis solution, mixed with SDS sample buffer, and loaded onto 8% acrylamide gels (10 ml). Gels were transferred to nitrocellulose membranes (0.45 mm, Biorad) and blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween 20 (TBS-T) for 1-2 h. Membranes were probed with antibodies for EV markers, anti-CD63 (1:1000, Abcam, ab59479), anti-Flotillin (1:1000, BD, 610821), and anti-Alix (1:1000 Sigma, SAB 4200476). As a negative control, anti-calnexin (1:1000, Cell Signaling, mAb 2679) was used. Appropriate secondary antibodies were added and detected by ECL (Thermo Scienti c, 32106 and 34095). The same procedure was applied to detect integrins and ECM proteins such as bronectin (Abcam, ab2413) and collagen (Abcam, ab34710).
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