For Western blot, neurons were washed three times with ice-cold PBS. Cell lysates were prepared in the modified RIPA buffer containing protease inhibitors (Thermo Scientific, Rockford, IL). Equal amounts of proteins, determined by BCA protein assay (Thermo Scientific, Rockford, IL), were loaded onto SDS-PAGE gel and immunoblotted using antibodies against clathrin heavy chain (1:1,000; BD Bioscience, San Jose, CA), dynamin (1:1,000; BD Bioscience, San Jose, CA, recognizing all dynamin isoforms, including 1, 2 and 3), dynamin 1 (1:1,000; Thermofisher, Waltham, MA) and β-actin (1:2,000; Abcam, Cambridge, United Kingdom).
Dynamin 1
Manufactured by Thermo Fisher Scientific
Dynamin 1 is a GTPase involved in membrane trafficking and endocytosis. It plays a key role in the formation of coated vesicles during clathrin-mediated endocytosis.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Dynamin, by BD (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
Hrp anti mouse, by Thermo Fisher Scientific (1 mentions)
Complete mini proteinase inhibitor mix, by Roche (1 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Luminata forte, by Merck Group (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
2 protocols using dynamin 1
1
Quantification of Clathrin and Dynamin
2
Western Blot Analysis of Protein Extracts
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Protein extracts were made in IP lysis buffer (10 mM HEPES pH 7.5, 137 mM NaCl, 0.4% NP-40, 10% glycerol) with Complete-mini proteinase inhibitor mix (Roche) added fresh. Extracts were quantified using the Bradford reagent (Bio-Rad). Extracts (50 μg protein) were diluted in Laemmli buffer, incubated at 95°C for 5 minutes, resolved by SDS-PAGE and transferred to nitrocellulose membrane. All membrane blotting steps were carried out in TBS plus Tween (TBST). Blots were blocked in 5% milk, then incubated at RT with primary antibody (for specific dilutions see below) for 4 hours, HRP-conjugated secondary antibody (1:15000) for 1h and visualized with Luminata Forte (Millipore). Membranes were incubated with Restore Western blot stripping buffer (Thermo Scientific) at room temperature for 10 minutes while shaking to remove antibodies for subsequent hybridization. Primary antibodies used were dynamin-1 (1:3000; Thermo Scientific, PA1-660), actin (1:1000; Abcam, ab3280). Secondary antibodies used were HRP anti-mouse (Thermo Scientific), HRP anti-rabbit (BioRad).
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