Tissue lysates were mixed with 4× sodium dodecyl sulfate (SDS) sample buffer, incubated at 100 °C for 5 min and run on Novex WedgeWell Tris-Glycine gels (4–20%, 8–16% or 10–20%) and transferred onto nitrocellulose membranes (Thermo Fisher Scientific) using mini-blot modules (Thermo Fisher Scientific). The membranes were then blocked in Pierce Protein-Free T20 (TBS) Blocking Buffer (Thermo Fisher Scientific) and incubated with the corresponding primary antibodies (Supplementary Table 2 ) overnight at 4 °C. Then, the membranes were washed in TBS with 0.1% Tween 20 three times and incubated with HRP-conjugated secondary antibodies (Supplementary Table 3 ) for 1 h. After three washes in TBS-Tween 20, the membranes were treated with SuperSignal West Pico or SuperSignal West Femto (Thermo Fisher Scientific) and imaged using an iBright CL1000 chemiluminiscence imaging system (Thermo Fisher Scientific).
Novex wedgewell tris glycine gels
Manufactured by Thermo Fisher Scientific
Novex WedgeWell Tris-Glycine gels are a type of polyacrylamide gel used for protein separation and analysis in electrophoresis techniques. They feature a wedge-shaped design that provides consistent and uniform resolution across the gel.
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Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Pierce protein free t20 tbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Mini blot module, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Protein ladder, by Thermo Fisher Scientific (1 mentions)
Mini gel tank, by Thermo Fisher Scientific (1 mentions)
Typhoon fla 9500 biomolecular imager, by GE Healthcare (1 mentions)
Powerpac basic, by Bio-Rad (1 mentions)
2 protocols using novex wedgewell tris glycine gels
1
Western Blot Analysis of Protein Expression
2
Benchmarking Protein Separation Techniques
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Protein samples were analyzed with polyacrylamide gels to benchmark performance of the microfluidic thermal gel separations. Samples were electrophoresed through Novex Wedge Well Tris-Glycine gels (6%, 10%, 16%, and 4-20%) (Thermo Fisher) using a Mini Gel Tank (Thermo Fisher) and a PowerPac Basic voltage supply (Bio-Rad, Hercules, CA). Proteins were prepared in PAGE native sample buffer and analyzed in PAGE running buffer (Thermo Fisher). The protein sample mixture contained 500 ng of each EGF (4.2 μM), ovalbumin (550 nM), and β-galactosidase (54 nM) and 100 ng of phycoerythrin (20 nM) to provide readily detectable signal for each protein band. A protein ladder (Thermo Fisher) was also analyzed on each gel. Gels were run for ~ 45 min at 4 °C to minimize Joule heating-induced band-broadening. Protein bands were imaged using a Typhoon FLA 9500 biomolecular imager (GE Healthcare Life Sciences, Piscataway, NJ).
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