Protein oxidation was determined using the OxyBlot™ kit, according to the manufacturer ’ s instructions. The method is based on immunochemical detection of protein carbonyl groups derivatized using 2,4-dinitrophenylhydrazine (DNPH). Briefly, spinal cord samples were reacted with DNPH and the samples were electrophoresed and electrotransfered to nitrocellulose membranes. Subsequently, the primary rabbit anti-DNP antibody (1:150) and horse-radish peroxidase-conjugated goat anti-rabbit antibody (1:300) were applied to detect protein carbonyl groups. Membranes were visualized by chemiluminescence using the Pierce SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). The bands were recorded using the Kodak Image Station CF 440 and the densitometric analyses of the proteins of all molecular weights were done using the Kodak 1D 3.5 imaging software (Eastman Kodak, Inc., Rochester, NY, USA).
1d 3.5 imaging software
Manufactured by Kodak
Sourced in United States
The Kodak 1D 3.5 imaging software is a tool designed for digital image processing and analysis. It provides basic functionalities for viewing, manipulating, and exporting digital images.
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Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Akt antibody, by Cell Signaling Technology (1 mentions)
Erk antibody, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti α sma antibody, by Santa Cruz Biotechnology (1 mentions)
2 protocols using 1d 3.5 imaging software
1
Detecting Protein Oxidation via OxyBlot
2
Western Blot Analysis of Protein Expression
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Equal amounts of proteins extracted from different groups of PAECs were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred to polyvinylidene difluoride membranes (EMD Millipore, Darmstadt, Germany). After blocking with 5% skimmed milk, the membranes were incubated with anti-α-SMA antibody (1 : 1000, Santa Cruz Biotechnology), ERK antibody (1 : 1000, Cell Signaling Technology), p-ERK antibody (Thr202/Tyr204, 1 : 2000, Cell Signaling Technology), Akt antibody (1 : 1000, Cell Signaling Technology), p-Akt antibody (Ser473, 1 : 2000, Cell Signaling Technology), GAPDH (1 : 1000, Cell Signaling Technology), and β-actin (1 : 1000, Abcam) overnight at 4°C. After washing three times, the membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibody (1 : 2000, Santa Cruz Biotechnology) and finally detected by enhanced chemiluminescence and analyzed using Kodak 1D 3.5 imaging software (Eastman Kodak, Rochester, NY, USA).
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