Protein extracts were made in lysis buffer (1mM HEPES, 137mMNaCl, 10% glycerol, 1% NP-40) with Complete-mini proteinase inhibitor mix (Roche) added fresh. Extracts were quantified using the Bradford reagent (Bio-Rad). Extracts (25–50 µg protein) were diluted in Laemmli buffer, incubated at 95°C for 5 minutes, resolved by SDS-PAGE and transferred to PVDF membrane. All membrane blotting steps were carried out in TBS plus Tween (TBST). Blots were blocked for 1 hour in 5% milk or 5% BSA, incubated at RT with primary antibody for 4 hours, HRP-conjugated secondary antibody (1:15000) for 1 hour and visualized with Luminata Forte (EMD Millipore). Membranes were incubated with Restore Western blot stripping buffer (Fisher) for 5 min while shaking to remove antibodies for subsequent hybridization. Primary antibodies used were rabbit anti-dynamin-1 (1:1500; Pierce Thermo Fisher, PA1-660), rabbit anti-DNM1a (1:200; Affinity BioReagents), chicken anti-DNM1b (1:100; Affinity BioReagents) and mouse anti-tubulin (1:1000; Sigma, T4026).
Complete mini proteinase inhibitor mix
Manufactured by Roche
Complete-mini proteinase inhibitor mix is a ready-to-use solution designed to inhibit a broad range of serine, cysteine, and metalloproteases. It can be used to prevent protein degradation during sample preparation and processing.
Automatically generated - may contain errors
Lab products found in correlation
Bradford reagent, by Bio-Rad (2 mentions)
Luminata forte, by Merck Group (2 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (2 mentions)
Mouse anti tubulin, by Merck Group (1 mentions)
Hrp anti mouse, by Thermo Fisher Scientific (1 mentions)
Dynamin 1, by Thermo Fisher Scientific (1 mentions)
2 protocols using complete mini proteinase inhibitor mix
1
Western Blot Analysis of Dynamin Proteins
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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