Denaturing Proteins for SDS-PAGE

Proteins for SDS-PAGE were denatured by heating to 95°C for 5 min in sample buffer (1% SDS, 5–15 mM DTT, bromphenol blue) and separated using the glycine/2-amino-2-methyl-1,3-propanediol/HCl (ammediol) system in 5–15 or 10–15% acrylamide gradient gels (Bury, 1981 (link)) casted in-house (10 × 10 × 0.15 cm). The gels were stained with Coomassie Brilliant Blue.

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Rasmussen C.B., Scavenius C., Thøgersen I.B., Harwood S.L., Larsen Ø., Bjerga G.E., Stougaard P., Enghild J.J, & Thøgersen M.S. (2023). Characterization of a novel cold-adapted intracellular serine protease from the extremophile Planococcus halocryophilus Or1. Frontiers in Microbiology, 14, 1121857.

Publication 2023

Acrylamide Ammediol Bromphenol blue Buffer Coomassie brilliant blue Gels Glycine Propanediol Proteins Sds page

Corresponding Organization : Aarhus University

Other organizations : Danish Technological Institute, NORCE Norwegian Research Centre

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Variable analysis

independent variables

Denaturation temperature (95°C)
Denaturation time (5 min)
SDS concentration (1%)
DTT concentration (5–15 mM)
Acrylamide gradient (5–15% or 10–15%)

dependent variables

Protein separation on SDS-PAGE
Protein visualization by Coomassie Brilliant Blue staining

control variables

Sample buffer composition (1% SDS, 5–15 mM DTT, bromphenol blue)
Electrophoresis system (glycine/2-amino-2-methyl-1,3-propanediol/HCl (ammediol))
Gel dimensions (10 × 10 × 0.15 cm)

controls

No positive or negative controls were explicitly mentioned in the input.

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