Proteins were resolved by 10% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad). Nonspecific binding sites were blocked with 5% (w/v) milk in TBS containing 0.1% Tween 20 (TBS-T). After blocking, blots were incubated overnight with the primary polyclonal antibody flotillin 1 (1:1000; Abcam), CD63 (1:200; Santa Cruz Biotechnology), TSG101 (1:1000; BD Biosciences), actinin-4 (1:1000; Gentex), microfilin (1:5000; Thermo Fisher Scientific), nSMase2 (1:500; Santa Cruz Biotechnology), transferrin receptor (1:1000; Invitrogen), and β-actin (1:5000; Sigma-Aldrich). After washes with TBS-T, blots were incubated for 2 hours with the appropriate IgG horseradish peroxidase–linked secondary antibody (1:1000; Cell Signaling Technology) and developed by enhanced chemiluminescence. Image analysis was performed using a G:BOX imaging system (Syngene).
Actinin 4
Manufactured by Gentex
Actinin-4 is a protein that is part of the alpha-actinin family. It is involved in regulating the structure and dynamics of the actin cytoskeleton within cells.
Automatically generated - may contain errors
Lab products found in correlation
Flotillin 1, by Abcam (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
G box imaging system, by Syngene (2 mentions)
Tsg101, by BD (2 mentions)
Igg horseradish peroxidase linked secondary antibody, by Cell Signaling Technology (2 mentions)
Transferrin receptor, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Nsmase2, by Santa Cruz Biotechnology (1 mentions)
Mitofilin, by Thermo Fisher Scientific (1 mentions)
2 protocols using actinin 4
1
Western Blot Analysis of Extracellular Vesicle Markers
2
Protein Quantification and Immunoblotting Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The protein amount in each fraction and pellet were measured by bicinchoninic acid protein assay. Equal amount of protein (20 μg) from each fraction and pellet were resolved by 10% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad; Hercules, CA). Nonspecific binding sites were blocked with 5% (w/v) milk in TBS containing 0.1% Tween 20 (TBS-T). After blocking, blots were incubated overnight with the primary polyclonal antibody flotillin 1 (1:1000; Abcam), CD63 (1:200; Santa Cruz Biotechnology), TSG101 (1:1000; BD Biosciences), actinin-4 (1:1000; Gentex), and mitofilin (1:5000; Thermo Fisher Scientific). After washes with TBS-T, blots were incubated for 2 h with the appropriate IgG horseradish peroxidase–linked secondary antibody (1:1000; Cell Signaling Technology) and developed by enhanced chemiluminescence. Image analysis was performed using a G: BOX imaging system (Syngene).
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