Samples were lysed with RIPA buffer (Thermo Fisher, #89900). Protein concentration was determined by bicinchonic acid assay. 100 µg of protein from each sample was loaded onto a Criterion 10% Tris-HCl gel (Bio-Rad, Hercules, CA, cat #3451018) and electrophoresed. The proteins were then transferred to a PVDF membrane (Bio-Rad, cat #1620174) and blocked with 5% powdered milk (Bio-Rad, cat #1706404) in Dulbecco’s PBS (Thermo Fisher, cat #14190250) +0.1%Tween®20 (Sigma-Aldrich, St. Louis, MO, cat #P9416) for 1 h. The membrane was subsequently incubated with mouse-anti-human CD63 primary antibody (BD, cat #556019), at a 1:1000 dilution for 1 hour, and mouse-anti-human CD81 (Santa Cruz Biotechnology, cat #sc-166029), at a 1:1000 dilution for 1 hour. After washing the membrane, it was incubated with a goat-anti-mouse IgG-HRP secondary antibody (Santa Cruz Technology, cat #sc-2005) at a 1:10,000 dilution for 1 h. The membrane was then incubated with a 1:1 mixture of SuperSignal West Pico Stable Peroxide solution (Thermo Fisher, cat #34080) and Luminol Enhancer solution (Thermo Fisher, cat #34080) for 5 min. The membrane was visualized on Amersham HyperfilmTM ECL chemiluminescence film (GE Life Sciences, PA, cat #28906839).
Mouse anti human cd63 primary antibody
Manufactured by BD
The Mouse-anti-human CD63 primary antibody is a laboratory reagent used to detect the presence of the CD63 protein in human samples. CD63 is a transmembrane glycoprotein commonly used as a marker for exosomes and other extracellular vesicles. This antibody can be used in various analytical techniques, such as Western blotting, flow cytometry, and immunohistochemistry, to identify and quantify CD63-positive cells or structures.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Dulbecco s pbs, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico stable peroxide solution, by Thermo Fisher Scientific (1 mentions)
Mouse anti human cd81, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg hrp secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Luminol enhancer solution, by Thermo Fisher Scientific (1 mentions)
Criterion 10 tris hcl gel, by Bio-Rad (1 mentions)
Tween 20, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab6709, by Abcam (1 mentions)
Wha1001325, by Merck Group (1 mentions)
2 protocols using mouse anti human cd63 primary antibody
1
Western Blot Analysis of Exosomal Markers
2
Immunogold Labeling of Extracellular Vesicles
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5 μl of EVs (from iodixanol fraction(s) of interest, immediately after the 250,000xg density gradient centrifugation) were adsorbed for 1 minute onto carbon-coated, standard thickness, 400 mesh support film grids (EMS CF400-CU) made hydrophilic by plasma treatment (glow discharge, 25 mA). Excess liquid was removed with Whatman grade 1 filter paper (Sigma-Aldrich WHA1001325), and grids were rinsed of phosphate and salts by floating briefly on a drop of water. For immunogold labelling, samples were blocked with 1% bovine serum albumin (BSA) for 10 minutes, and incubated with mouse anti-human CD63 primary antibody (BD Pharmingen 556019, 1:20 in 1% BSA) for 30 minutes at room temperature. Grids were washed with 3 drops of PBS-/-in 10 minutes, then stained with rabbit anti-mouse bridging antibody (Abcam ab6709, 1:50 in 1% BSA) for 10 minutes. After further rinsing, grids were incubated with 10 nm Protein A-gold particles (University Medical Center Utrecht,1:50 in 1% BSA) for 20 minutes. Samples were then washed in PBS-/-(2 changes in 5 minutes) and water (4 changes in 10 minutes). Grids were blotted again, then stained with 0.75% uranyl formate (EMS 22451) for 15 seconds. Excess uranyl formate was then removed by blotting, and grids were examined under a transmission electron microscope (JEOL 1200EX) and imaged with a CCD camera (AMT 2K).
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