Horseradish peroxidase streptavidin (SA-5004) was from Vector Laboratories (Burlingame, CA, USA). SuperSignal West Pico chemiluminescent substrate was from Thermo Scientific (Rockford, IL, USA). Myoglobin from equine skeletal muscle (M0630) was from Sigma-Aldrich. N-Homocysteinylated myoglobin was prepared as we previously reported [64 (link)]. Biotinyl-Asp-Glu-Val-Asp-aldehyde (CAS registry number: 178603-73-1) was from Bachem Americas (N-1470, Torrance, CA, USA; see Figure S1 for its structure in Support Information ). Rhodamine-aldehyde (10 mM) in 50% 200-proof ethanol was synthesized as previously described [64 (link)]. EZ-Run prestained protein ladder (BP3603) was from Fisher Scientific. Natural unstained protein ladder (161-0317) was from Bio-Rad. Biotinylated protein markers were from Sigma-Aldrich (B2787) and Bio-Rad (161-0319), respectively. All reagents were ACS grade and used as received without further purification. All incubations were carried out in an Eppendorf Thermomixer® (Eppendorf North America, Hauppauge, NY, USA) at 25 °C unless specified otherwise.
Protocol detail
Homocysteinylated Myoglobin Labeling Protocol
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Horseradish peroxidase streptavidin (SA-5004)
SuperSignal West Pico chemiluminescent substrate
M0630)
Biotinyl-Asp-Glu-Val-Asp-aldehyde
EZ-Run prestained protein ladder (BP3603)
Natural unstained protein ladder (161-0317)
B2787
Thermomixer
Aldehyde
Asp asp
Equine
Ethanol
Horseradish peroxidase
Myoglobin
Protein
Rhodamine
Skeletal muscle
Streptavidin
Vector
Corresponding Organization : Wenzhou Medical University
Other organizations : Tufts University, Harvard University, Brigham and Women's Hospital, Nova Southeastern University
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Variable analysis
independent variables
- Biotinyl-Asp-Glu-Val-Asp-aldehyde
- Rhodamine-aldehyde
- N-Homocysteinylated myoglobin
- Horseradish peroxidase streptavidin (SA-5004)
- SuperSignal West Pico chemiluminescent substrate
- Myoglobin from equine skeletal muscle (M0630)
- EZ-Run prestained protein ladder (BP3603)
- Natural unstained protein ladder (161-0317)
- Biotinylated protein markers
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