Dorsal skin tissues were homogenised in radioimmunoprecipitation assay (RIPA) buffer. After centrifugation, supernatants were collected and total protein concentration was measured using BCA Protein Assay Kits (Thermo Scientific, Waltham, MA, USA). An equal amount of proteins was resolved in 10–12% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes using a semidry transfer system (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% non-fat milk in 0.1% Tween 20 in phosphate buffered saline and hybridised with appropriate primary antibodies overnight. The membranes were then incubated with horseradish peroxidase–conjugated secondary antibodies for 3 h. Finally, protein bands were detected using enhanced chemiluminescence western blotting reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Enhanced chemiluminescence western blotting reagent
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Enhanced chemiluminescence western blotting reagents are a set of laboratory tools used for the detection and analysis of proteins in Western blotting experiments. These reagents are designed to produce a luminescent signal when interacting with the target protein, allowing for its visualization and quantification.
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5 protocols using enhanced chemiluminescence western blotting reagent
1
Western Blot Analysis of Protein Extracts
2
Western Blot Analysis of Skin Proteins
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Skin tissues were homogenized in cold RIPA buffer with protease inhibitor cocktail. Homogenates were centrifuged at 16,000× g for 10 min at 4 °C to obtain supernatants. To determine protein expression, an equal the mount proteins were separated by 10–12% SDS-PAGE and then transferred to polyvinylidene fluoride membrane using a semidry transfer system (Bio-Rad, Hercules, CA, USA). Membranes were incubated with the appropriate primary antibodies overnight and with horseradish peroxidase-conjugated secondary antibodies for 3 h. Finally, protein bands were visualized using enhanced chemiluminescence western blotting reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
3
Protein Expression Analysis in Hair Cells
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Proteins were extracted from skin tissues or cultured (human hair dermal papilla cells) HHDPCs by homogenizing cells with radioimmunoprecipitation assay buffer. The protein content of each sample was measured using a BCA protein assay kits (Pierce Manufacturing, Appleton, WI, USA). To analyze protein expression, an equal amount of proteins from each group were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene fluoride membranes using a semidry transfer system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked with 5% non-fat milk and then hybridized with proper primary antibodies overnight at 4°C. After washing with 0.1% Tween-20 in phosphate-buffered saline, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 3 h at 4°C. Protein bands were visualized using enhanced chemiluminescence Western blotting reagents (Santa Cruz Biotechnology, Inc.).
4
Quantitative Western Blot Analysis
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Total protein from each skin specimen was extracted using RIPA Buffer (Cell Signaling Technology) following the manufacturer’s instructions. The protein content was quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were separated on SDS-PAGE gels and subsequently electro-blotted onto Immobilon-P polyvinylidene fluoride membrane (Millipore) using a semidry transfer system (Bio-Rad). The membranes were blocked with 5% skim milk and then incubated with the appropriate primary antibodies overnight at 4°C. After washing, the membranes were hybridized with horseradish peroxidase-conjugated secondary antibodies for 3 h. The protein blots were visualized using an enhanced chemiluminescence Western blotting reagent (Santa Cruz Biotechnology).
5
Protein Extraction and Western Blotting Protocol
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Cells and liver tissues were homogenized in RIPA buffer and then centrifuged at 10,000× g, 4 °C for 10 min to obtain supernatant. Total protein concentration of each sample was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Equal amounts of protein were resolved on SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA) using a semidry transfer system (Bio-rad, Hercules, CA, USA). After blocking with 5% non-fat milk in PBST (0.1% Tween 20 in PBS) for 2 h at 4 °C, the membranes were incubated overnight with appropriate primary antibodies at 4 °C. Then, the membranes were washed five times with PBST and hybridized with horseradish peroxidase-conjugated anti-rabbit or anti-goat immunoglobulin IgG secondary antibodies for 3 h at 4 °C. After washing five times with PBST, blots were visualized using enhanced chemiluminescence western blotting reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
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