Protein lysats of cells, AB and MV were prepared in RIPA buffer containing a cocktail of protease inhibitors (Sigma). Proteins were denatured in Laemmli buffer and separated by 4-12% gradient SDS-PAGE in non-reducing (tetraspanins CD81, CD63, CD9) or reducing (all other) conditions and transferred to a nitrocellulose membrane (CD63, flotillin-1, peIF2a and CHOP; Fisher Scientific) or PVDF membrane (all other; BIO-RAD, Marnes La Coquette, France). Membranes were incubated with primary antibodies CD63 (NVG-2; 1:1000; BioLegend), CD81 (Eat-2; 1:1000; BioLegend), CD9 (EM-04; 1:1000; Abcam, Cambridge, UK), polyclonal rabbit anti-calnexin antibody (1:1000; Euromedex, Souffelweyersheim, France), β–actin (W16197A; 1:20,000; Biolegend), flotillin-1 (W16108A; 1:1000; Biolegend), peIF2a (D9G8; 1:1000; Ozyme) and CHOP (D46F1; 1:1000; Ozyme) blocked by either TBS 0.05% Tween-20 4% BSA (CD81, peIF2a, CHOP) or TBS 0.05% Tween-20 5% milk (CD9, CD63, calnexin, flotillin-1), followed by incubation with cognate HRP-conjugated secondary antibodies 1:100,000. The signals were detected with enhanced chemiluminescence substrate (ECL West Pico Femto, Fischer Scientific) on a Fusion FX6 instrument (Fisher Scientific).
Cd81 eat 2
Manufactured by BioLegend
Sourced in United Kingdom
CD81 (Eat-2) is a cell surface protein that belongs to the tetraspanin family. It functions as a molecular organizer, facilitating the assembly and regulation of multimeric protein complexes on the cell surface.
Automatically generated - may contain errors
Lab products found in correlation
Peif2a, by Thermo Fisher Scientific (2 mentions)
Cocktail of protease inhibitor, by Merck Group (2 mentions)
Peif2a, by Ozyme (2 mentions)
Flotillin 1, by Ozyme (2 mentions)
Cd63 nvg 2, by BioLegend (2 mentions)
Cd9 em 04, by Abcam (2 mentions)
β actin w16197a, by BioLegend (2 mentions)
Ecl west pico femto, by Thermo Fisher Scientific (2 mentions)
Fusion fx6 instrument, by Thermo Fisher Scientific (2 mentions)
Flotillin 1, by Thermo Fisher Scientific (2 mentions)
Cd11c pe, by BD (1 mentions)
Cd71 pe, by BD (1 mentions)
Cd69 pe, by BD (1 mentions)
Cd25 apc, by BD (1 mentions)
Cd45 pe cy7, by BD (1 mentions)
Epcam apc, by BD (1 mentions)
Cd11c pe cy7, by BD (1 mentions)
Cd69 pe cy7, by BD (1 mentions)
B220 biotin, by BD (1 mentions)
Foxp3 pe, by BD (1 mentions)
3 protocols using cd81 eat 2
1
Western Blot Analysis of Cell Lysates
2
Immunophenotyping and Protein Analysis
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The following antibodies were purchased from BioLegend, eBioscience, or BD Biosciences and used for flow cytometry or enrichment: FITC-Sirpα, PE/Cy7-Sirpα, PerCP/Cy5.5-B220, APC-EpCAM, PE-CD11c, PE/Cy7-CD11c, Brilliant Violet 605-CD11c, Pacific Blue-IA/IE, FITC-CD8α, Alexa700-CD8α, PE-Vα2, APC-Vα2, APC-Vβ5, PE-Vβ6, PE-Foxp3, Pacific Blue-Foxp3, PerCP/Cy5.5-Thy1.1, APC-TCRβ, PE-CD69, PE/Cy7-CD69, Brilliant Violet 605-CD4, APC-CD25, Brilliant Violet 605-CD25, Brilliant Violet 785-CD25, FITC-CD45RB, APC-ICAM1, PR-CD9, PE-CD81, APC-CD48, PE/Cy7-CD24, APC-CD24, PE-CD71, PE/Cy7-CD45, APC-streptavidin, Biotin-Thy1.2, Biotin-CD8β, Biotin-CD25, and Biotin-B220. Alexa647-CTxB subunit was purchased from Molecular Probes. For Western blotting, the following clones were used: MHCII β chain (Bett; homemade), CD48 (OX-78; Serotec), CD24 (M1/69; BioLegend), actin (A2066; Sigma), CD9 (LMC8; eBioscience), and CD81 (Eat2; BioLegend), and HRP-conjugated secondary antibodies (anti-rat, anti-rabbit, and anti-hamster) were purchased from Jackson ImmunoResearch or BIOSOURCE. Myriocin, MβCD, cholesterol, sphingomyelin, and 4-hydroxy-tamoxifen were purchased from Sigma.
3
Western Blot Analysis of Tetraspanins
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Protein lysats of cells, AB and MV were prepared in RIPA buffer containing a cocktail of protease inhibitors (Sigma). Proteins were denatured in Laemmli buffer and separated by 4-12% gradient SDS-PAGE in non-reducing (tetraspanins CD81, CD63, CD9) or reducing (all other) conditions and transferred to a nitrocellulose membrane (CD63, flotillin-1, peIF2a and CHOP; Fisher Scientific) or PVDF membrane (all other; BIO-RAD, Marnes La Coquette, France). Membranes were incubated with primary antibodies CD63 (NVG-2; 1:1000; BioLegend), CD81 (Eat-2; 1:1000; BioLegend), CD9 (EM-04; 1:1000; Abcam, Cambridge, UK), polyclonal rabbit anti-calnexin antibody (1:1000; Euromedex, Souffelweyersheim, France), β-actin (W16197A; 1:20,000; Biolegend), flotillin-1 (W16108A; 1:1000; Biolegend), peIF2a (D9G8; 1:1000; Ozyme) and CHOP (D46F1; 1:1000; Ozyme) blocked by either TBS 0.05% Tween-20 4% BSA (CD81, peIF2a, CHOP) or TBS 0.05% Tween-20 5% milk (CD9, CD63, calnexin, flotillin-1), followed by incubation with cognate HRP-conjugated secondary antibodies 1:100,000. The signals were detected with enhanced chemiluminescence substrate (ECL West Pico Femto, Fischer Scientific) on a Fusion FX6 instrument (Fisher Scientific).
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