A total of 20 μg of proteins was separated by SDS‐PAGE and then transferred to PDVF membrane. The membrane was blocked with 5% BSA or 5% non‐fat dry milk, then incubated with primary antibody and HRP‐conjugated secondary antibody (Table S1 ). Separated proteins were detected by Clarity Western ECL Substrate (Bio‐Rad laboratories, Hercules, CA, USA) and Las‐4000 (Bio‐Rad Laboratories), and were analyzed using Image‐J software (version 1.50i). Details are described in the (Doc. S3 ).
Las 4000
Manufactured by Bio-Rad
Sourced in United States
The Las-4000 is a high-performance imaging system designed for the analysis of fluorescent and chemiluminescent signals in a variety of life science applications. It features a large imaging area, a sensitive CCD camera, and user-friendly software for capturing and analyzing images.
Automatically generated - may contain errors
Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Ezripa lysis kit, by Takara Bio (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Nitrocellulose blotting membrane, by GE Healthcare (1 mentions)
Tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Multi gauge v3, by Fujifilm (1 mentions)
4 protocols using las 4000
1
Western Blot Protein Detection
2
Western Blot Protein Detection Protocol
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Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Ishikawa et al. 2014 (link)). The membranes were blocked with 5% nonfat dried milk in TBST (Tris-buffered saline containing 0.1% [w/v] Tween 20) for 1 h and then incubated with their respective antibodies for 1 h at room temperature. The membranes were then washed three times with TBST for 10 min, incubated with a secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature, and washed three times with TBST for 10 min. Clarity Western ECL substrate (Bio-Rad) was used for detection with an LAS4000.
3
Western Blotting Analysis of Cell Lysates
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Cells and skin tissues were lysed using radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (EzRIPA lysis kit; TaKaRa, Tokyo, Japan). After sonication, samples were centrifuged at 14,000× g for 15 min at 4 °C. Protein concentration was determined using the bicinchoninic acid assay (Thermo Fisher Scientific). Equal amounts of protein were separated using 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes previously activated with methanol. PVDF membranes were blocked with 5% skim milk at room temperature to prevent the binding of non-specific proteins. Membranes were then incubated with appropriately diluted primary antibodies (Table S1 ) at 4 °C overnight. Membranes were rinsed with tris-buffered saline containing 0.1% Tween 20 and incubated with secondary antibodies at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence solution (Cytiva) using LAS-4000 (Bio-Rad, Hercules, CA, USA). Individual protein expression values were quantified using Image J software (National Institutes of Health, NIH, Maryland, MD, USA) and normalized to beta-actin (Cell Signaling Technology) to control the differences in protein loading. Values for a single blot are expressed relative to the mean of the first group.
4
Protein Extraction and Western Blot Analysis
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Lesion segments ( 1 cm in length) were extracted using a tissue protein extraction reagent (Thermo Fisher Scientific) with a protease inhibitor cocktail tablet (Roche) and 1 mM phenylmethylsulphonyl fluoride (PMSF). The amount of extracted protein was then quantified using bicinchoninic acid protein quantitative analysis. Proteins (40 mg/mL) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose blotting membrane (GE Healthcare, Freiburg, Germany). Blotting membranes were blocked using 3% BSA in TBST for 1 h at RT and incubated overnight at 4 ℃ with the primary antibodies. The membranes were washed and incubated with secondary peroxidase-conjugated antibodies for 1 h at RT on the following day. Protein expression was confirmed using enhanced chemiluminescence solution, which was exposed to LAS 4000 (BioRad, Hercules, CA, USA). Quantification of band intensity was performed using a multi-gauge V 3.0 software (Fujifilm, Tokyo, Japan). Full-length western blot images are presented in Additional file 7: Fig. S7 .
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