The antibodies utilized were antigen affinity-purified polyclonal sheep IgG anti-rabbit CD81 (Catalog no. 0349509; BioLegend, San Diego, California, USA) and antigen affinity-purified polyclonal IgG anti-rabbit CD83 (Catalog no. MBS127731, MyBioSource, Inc., San Diego, California, USA). Protein was extracted from isolated exosomes by utilizing radio-immuno-precipitation buffer's composition. Twenty nanograms of protein were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 4~20% polyacrylamide gradient gels. After being incubated in 5% non-fat dry milk, Tris hydrochloride, 0.1% Tween 20 for an hour, primary antibodies (1:500 dilution factor for all target proteins) were added to one of the membranes including specimen samples and incubated at 4 °C overnight. HRP-conjugated secondary antibody (Goat anti-rabbit IgG-HRP-1mg Goat mab -Novus Biologicals) solution was incubated against the blotted target protein for two hours at room temperature. Following washing for six times in 1 X TBS-T, densitometric analysis of the immunoblots was done to quantify the amounts of CD83 and CD81 against housekeeping protein β-actin by image analysis software on the Chemi Doc MP imaging system (version 3) generated by Bio-Rad [18 ]. This was performed at the Biochemistry department at faculty of Medicine, Cairo university.
Polyclonal sheep igg anti rabbit cd81
Manufactured by BioLegend
Sourced in United States
Polyclonal sheep IgG anti-rabbit CD81 is a laboratory reagent used to detect and quantify the expression of the CD81 protein in rabbit samples. It is a polyclonal antibody produced in sheep and specifically targets the CD81 antigen.
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2 protocols using polyclonal sheep igg anti rabbit cd81
1
Exosomal Protein Quantification by Western Blot
2
Exosomal Protein Expression Analysis
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Exosomes were lysed in RIPA buffer using protease inhibitors and 1 mM phenylmethylsulfonyl fluoride. Exosomal lysates in equal volumes (50 μL) were subjected to non-reducing 12.5% SDS-PAGE for CD63 and CD81 and subsequently transferred utilizing a wet transfer system (Bio-Rad Laboratories, Hercules, CA, USA) on a PVDF membrane (MDI Membrane Technologies, Harrisburg, PA, USA). After blocking, in 1 TBS-T solution (1:5000; Abcam, Cambridge, MA, USA) containing 5 percent nonfat skim milk, the membrane was incubated with the CD63 primary antibody. The antibodies utilized were CD63 (Biolegend, Cat. No. 0353007) and polyclonal sheep IgG anti-rabbit CD81 (Biolegend, Cat. No. 0349509) with antigen affinity purification, as well as beta-actin. They were incubated at 4°C overnight. After being washed, the blot was incubated with a secondary antibody conjugated to horseradish peroxidase (HRP), and it was then developed utilizing an ECL imager (Invitrogen, a brand of Thermo Fisher Scientific).
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