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).
Igg horseradish peroxidase linked secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The IgG horseradish peroxidase–linked secondary antibody is a laboratory reagent used to detect and quantify the presence of primary antibodies in various immunoassays. It consists of an anti-IgG antibody conjugated to the enzyme horseradish peroxidase, which can catalyze a colorimetric or chemiluminescent reaction for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
G box imaging system, by Syngene (4 mentions)
Flotillin 1, by Abcam (3 mentions)
Tsg101, by BD (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
Actinin 4, by Gentex (2 mentions)
Mitofilin, by Thermo Fisher Scientific (2 mentions)
Transferrin receptor, by Thermo Fisher Scientific (1 mentions)
Nsmase2, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
4 protocols using igg horseradish peroxidase linked secondary antibody
1
Western Blot Analysis of Extracellular Vesicle Markers
2
Protein Quantification and Immunoblotting Analysis
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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).
3
Confirming EV Presence via Western Blot
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Western blotting was used to confirm the presence of EVs in our samples. Briefly, proteins were resolved by 8–16% Precise Protein Gel SDS–polyacrylamide (Thermo Fisher Scientific, Abingdon, UK) electrophoresis and transferred using a semi-dry system to polyvinylidene difluoride membranes (PVDF) (Bio-Rad, Hemel Hempstead, UK). After blocking, blots were incubated overnight with the primary polyclonal antibody TSG101 (1:250; 5% bovine serum albumin, 4A10 Ab83 Abcam, Cambridge, UK) (marker of presence of EVs). Detection was performed with the appropriate IgG horseradish peroxidase–linked secondary antibody (1:5000; Cell Signaling Technology Danvers, MA, USA). Image analysis was performed using a G:BOX imaging system (Syngene, Cambridge, UK).
4
Exosome Protein Profiling by Western Blot
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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 antibodies to GFP (1:1000; Thermo Fisher) CD63 (1:200; Santa Cruz Biotechnology), flotillin1 (1:1000; Abcam), TSG101 (1:1000; BD Biosciences), mitofilin (1:5000 Thermo Fisher Scientific) and α-actinin (1:1000; Gentex). After washes with TBS-T, blots were incubated for 2 h with the corresponding 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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