In vitro degradation assays were performed as published previously (Jonas et al., 2013 (link)). The reaction was carried out in Lon reaction buffer (25 mM Tris pH 8.0, 100 mM KCl, 10 mM MgCl2, and 1 mM DTT) employing 0.75 µM Lon (0.125 µM Lon6), 4 µM substrate (if not stated otherwise), and an ATP regeneration system (4 mM ATP, 15 mM creatine phosphate, and 75 µg/ml creatine kinase). The reaction and the ATP regeneration system were prepared separately pre-warmed to 30°C (approx. 4 min). The reaction was started by adding the ATP regeneration system. Samples were taken at indicated time points and quenched by 1 vol. 2× SDS sample buffer (120 mM Tris-Cl pH 6.8, 4% SDS, 20% glycerol, and 0.02% bromophenol blue) and snap frozen in liquid nitrogen. Samples were boiled at 65°C for 10 min and separated by SDS-Page (Bio-Rad 4–20% Mini-PROTEAN TGX Stain-Free Protein Gel), visualized by InstantBlue Protein Stain (Expedeon) and quantified using Bio-Rad ImageLab 6.0.1. Substrate levels were normalized to Lon and/or creatine kinase levels (in case of ‘–Lon’ samples).
Mini protean tgx stain free protein gel
Manufactured by Abcam
The 4–20% Mini-PROTEAN TGX Stain-Free Protein Gel is a pre-cast polyacrylamide gel designed for the separation and visualization of proteins. It features a 4-20% gradient that allows for the effective separation of a wide range of protein molecular weights. The gel is formulated with a stain-free technology that enables direct visualization of proteins without the need for additional staining steps.
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2 protocols using mini protean tgx stain free protein gel
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In vitro Lon Protease Degradation Assay
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In vitro Lon Protease Degradation Assay
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In vitro degradation assays were performed as published previously [24] (link). The reaction was carried out in Lon reaction buffer (25 mM Tris pH8.0, 100 mM KCl, 10 mM MgCl2, 1 mM DTT) employing 0.75 µM Lon (0.125 µM Lon6), 4 µM substrate (if not stated otherwise) and an ATP regeneration system (4 mM ATP, 15 mM creatine phosphate, 75 µg/ml creatine kinase). The reaction and the ATP regeneration system were prepared separately pre-warmed to 30 °C (approx. 4 min). The reaction was started by adding the ATP regeneration system. Samples were taken at indicated time points and quenched by 1 vol. 2x SDS sample buffer (120 mM Tris-Cl pH 6.8, 4 % SDS, 20 % glycerol, 0.02 % bromophenol blue) and snap frozen in liquid nitrogen. Samples were boiled at 65 °C for 10 minutes and separated by SDS-Page (Bio-Rad 4-20 % Mini-PROTEAN® TGX Stain-Free™ protein gel), visualized by InstantBlue protein stain (Expedeon) and quantified using Bio-Rad ImageLab 6.0.1. Substrate levels were normalized to Lon and/or creatine kinase levels (in case of -Lon samples).
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