Tissue specimens were lysed in a lysis buffer (100 mmol/L dithiothreitol, 50 mmol/L Tris-HCl, pH 6.8, 2% SDS, and 10% glycerol) containing protease inhibitors. Total protein concentration was assessed via the BCA method. The total protein was kept at −80°C awaiting subsequent analysis. Equal amounts of proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technology and transferred to PVDF (polyvinylidene fluoride) membrane. 5% diluted skimmed milk powder was used as a sealant and imprinted in Tris-Buffer salt water for 3 h at a time. Rabbit anti-substance P (dilution: 1:500, GeneTex, Inc., United States) and mouse anti-c-fos (dilution: 1:200, Santa Cruz Biotechnology, United States) monoclonal antibodies were blotted at 4°C for overnight incubation. After washing, the membrane was incubated with conjugated goat anti-mouse IgG (1:8000) or goat anti-rabbit IgG (1:5000) for 2h at room temperature. Next, the membrane was washed with buffer. Immune response bands were examined using the ChemiDoc XRS + Bio-Rad imaging system. Image J software (National Institutes of Health) was applied to determine the quantitative density of the membrane, with beta-actin (dilution: 1:1000, Santa Cruz Biotechnology, United States) as the control.
Rabbit anti substance p
Manufactured by GeneTex
Sourced in United States
Rabbit anti-substance P is a primary antibody that specifically binds to the neuropeptide substance P. It is intended for use in research applications such as immunohistochemistry, western blotting, and ELISA to detect and quantify substance P in biological samples.
Automatically generated - may contain errors
2 protocols using rabbit anti substance p
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of Substance P and c-Fos
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The specimens were lysed with lysis buffer (100 mmol/L dithiothreitol, 50 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, and 10% glycerol) containing protease inhibitors (Roche). The bicinchoninic acid assay was used to determine total protein concentration. Equal amounts of protein were resolved on a sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk in TBST buffer for 1 hour. The membranes were washed 3 times with TBST buffer and then incubated with monoclonal antibodies against rabbit antisubstance P (dilution: 1:400, GeneTex, Inc.) and mouse anti-c-fos (dilution: 1:200, Santa Cruz Biotechnology) at room temperature for 1.5 hours. After an additional 3 washes, the samples were incubated for 2 hours at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG (1:1000) or goat anti-rabbit IgG (1:1000). The membranes were then washed with TBST buffer, and the images were scanned with a GS800 Densitometer Scanner. The optical density data were analyzed using Image-Pro Plus software (Media Cybernetics). We used beta-actin (dilution: 1:1000, Santa Cruz Biotechnology) as a control.
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