Transfection of HEK293T cells was performed with EGFP-tauWT and mCherry or CCT5-mCherry or EGFP-tauP301L for 48 h. The cells were harvested in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100 supplemented with protease inhibitor mixture as above) on ice. Native electrophoresis was performed at neutral pH 7.5 using the NativePAGE Novex 4–16% Bis-Tris Gel system (Life Technologies) in which G-250 was used as a negative-charge shift molecule maintaining the proteins in their native state. Gels were run at 4 °C at 150 V.
Nativepage novex 4 16 bis tris gel system
Manufactured by Thermo Fisher Scientific
The NativePAGE Novex 4-16% Bis-Tris Gel System is a laboratory equipment used for the analysis of native proteins. It is designed to separate and analyze proteins in their native, non-denatured state, preserving their structure and function. The system includes pre-cast polyacrylamide gels, buffers, and accessories necessary for the electrophoresis process.
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3 protocols using nativepage novex 4 16 bis tris gel system
1
Native Electrophoresis of EGFP-tau Complexes
2
Native PAGE Protein Separation Protocol
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The Life Technologies NativePAGE Novex 4-16% Bis-Tris Gel System was used in accordance with the manufacturer’s protocol. Samples were prepared in a 30 μl total volume containing 7.5 μL of NuPage LDS sample buffer (4×), 3 μl of 5% G-250 sample additive and 1.5 μl of DDM 10% (n-dodecyl-β-D-maltoside). The NativeMark™ unstained protein standard (Life Technologies, Carlsbad, CA, USA) was used as a protein ladder.
3
Native PAGE Analysis of Protein Size
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For analysis of protein molecular size under native conditions, the Life Technologies NativePAGE Novex 4-16% Bis-Tris Gel System was used in accordance with the manufacturer's protocol. Samples were prepared in a 30 µL total volume containing 7.5 μL of NuPage LDS sample buffer (4×), 3 µl of 5% G-250 sample additive and 1.5 µl of DDM 10% (n-dodecyl-β-D-maltoside). The NativeMark™ unstained protein standard (Life Technologies, Carlsbad, CA, USA) was used as a protein ladder.
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