For the western blot analysis, exosomal proteins were isolated from 500 μl of plasma (the yield of exosomal protein was 9 mg/sample). The exosomal pellet was resuspended in RIPA buffer (Cell Signaling technology), supplemented with phosphatase and protease inhibitors (Roche), and incubated in ice for 20′. Samples were then harvested at 14,000 x g for 10′, and the supernatant collected in a new eppendorf. Protein concentration was determined by using Bradford Assay (Bio-Rad), following the manufacturer’s instructions. 80 μg of exosomal lysate were then loaded on a Criterion Tris-HCl 4–20% pre-cast gel (Bio-Rad), transferred onto a nitrocellulose membrane (Bio-Rad) and probed with anti-Alix (1:1000), anti-TSG101 (1:1000), anti-Calnexin (Sigma) (1:2000), and anti-CD9 (Cell Signaling Technology) (1:1000) primary antibodies, followed by isotype matched, horseradish-peroxidase-conjugated secondary antibodies. Finally, the proteins of interest were detected through chemi-luminescence reaction.
Anti alix
Manufactured by Merck Group
Sourced in United Kingdom
Anti-Alix is a laboratory equipment product. It is used for the detection and quantification of the Alix protein. Alix is a protein involved in various cellular processes. Anti-Alix is designed to assist researchers and scientists in their studies related to Alix.
Automatically generated - may contain errors
Lab products found in correlation
Anti tsg101, by Merck Group (2 mentions)
Criterion tris hcl 4 20 pre cast gel, by Bio-Rad (1 mentions)
Anti calnexin, by Merck Group (1 mentions)
Anti cd9, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Silverxpress kit, by Thermo Fisher Scientific (1 mentions)
Plusone silver stain kit, by GE Healthcare (1 mentions)
Anti eef1a, by Merck Group (1 mentions)
Anti vsv g, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
2 protocols using anti alix
1
Exosomal Protein Analysis by Western Blot
2
Protein Separation and Detection
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Proteins were separated on sodiumdodecyl sulfate (SDS)-polyacrylamide gels (10% acrylamide) and either visualised by silver staining using the Silver Stain PlusOne kit (GE Healthcare, Little Chalfont, UK) or SilverXpress kit (Invitrogen) or transferred to nitrocellulose membranes (Hybond enhanced chemiluminescence, ECL, Amersham). Antibodies used were: anti-p24 (1:100, mouse monoclonal, ARP 365, CFAR, NIBSC, UK; binds sequence NPPIPVGEIY in p24 of HIV-1 Gag), anti-VSV-G (1:2000, mouse monoclonal, Sigma-Aldrich), anti-RDpro pg70 (1:2000, goat polyclonal, Quality Biotech Inc., Camden, NJ, USA), anti-AHANAK (1:500, mouse-monoclonal, Abcam, Cambridge, UK), anti-TSG101 (1:500, rabbit-polyclonal, Sigma-Aldrich), anti-ALIX (1:1000, rabbit polyclonal), anti-EEF1A (1:500, mouse-monoclonal), anti-ENO1 (1:500, mouse-monoclonal), anti-MARCKSL1 (1:1000, rabbit-polyclonal) and anti-GAPDH (1:5000, mouse-monoclonal, all by Millipore, Billerica, MA, USA).
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