Protein was extracted using RIPA buffer (0.1% SDS, 1% Nonidet P‐40, 150 mmol/L NaCl, 0.5% sodium deoxycholate, 50 mmol/L Tris and protease inhibitor mixture; Roche). Protein concentrations were measured with the enhanced BCA Protein Assay Kit (Proteintech Group Inc). Samples (50 μg) were subjected to SDS‐PAGE with 10% polyacrylamide gel followed by electrotransfer into polyvinylidenedifluoride membranes and then blocked with Tris‐buffered saline (TBS), containing 5% non‐fat dry milk for 1 hour. The membranes were incubated with primary antibodies overnight at 4°C, washed with TBS three times and then incubated with the secondary antibody at room temperature for 1 hour. The Odyssey Infrared Imaging System (Li‐COR Biosciences) was used to visualize the bands, and Quantity One image analysis software was used to analyse the relative intensities of the protein bands. GAPDH was used to normalize the densitometric intensity for proteins. Antibodies used were as following: anti‐Ki67 (MA5‐14520, Thermo Fisher Scientific), anti‐PH3 (PA5‐17869, Thermo Fisher Scientific), anti‐Aurora B (ab2254, Abcam), anti‐YAP (ab205270, Abcam) and anti‐GAPDH (ab8245, Abcam).
Enhanced bca protein assay kit
Manufactured by Proteintech
Sourced in China
The Enhanced BCA protein assay kit is a colorimetric assay designed to quantify total protein concentration. It utilizes a modified bicinchoninic acid (BCA) reaction to produce a purple-colored complex, which can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Ab2254, by Abcam (1 mentions)
Ab205270, by Abcam (1 mentions)
Ma5 14520, by Thermo Fisher Scientific (1 mentions)
Pa5 17869, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ab8245, by Abcam (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Anti il10, by ABclonal (1 mentions)
Anti cd86, by Affinity Biosciences (1 mentions)
Anti ho1, by Huabio (1 mentions)
Anti inos, by Affinity Biosciences (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti nrf2, by Proteintech (1 mentions)
Enhanced chemiluminescence, by Abcam (1 mentions)
Anti interleukin il 1β, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Anti il 10, by Abcam (1 mentions)
Anti tnf α, by Santa Cruz Biotechnology (1 mentions)
3 protocols using enhanced bca protein assay kit
1
Western Blot Analysis of Cell Proliferation Markers
2
Protein Expression Analysis in Mouse Spinal Cord
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Following the experimental procedures, mice were deeply anesthetized and sacrificed. Spinal dorsal horn (L3-L5) tissues were promptly dissected, and RIPA lysis buffer (Beyotime, China) along with protease inhibitors got supplemented. Total protein was extracted using the lysis buffer. After centrifugation at 12,000 g and 4°C for 30 min, subsequently harvesting supernatant. We detected protein concentration utilizing an enhanced BCA protein assay kit (Proteintech, Wuhan, China). Equivalent amounts of protein got loaded onto 10‑15% percent SDS-PAGE gel for electrophoresis gel for different protein detections, subsequently transferred onto PVDF membranes (Millipore, MA). Submerging PVDF membranes with 5% milk for 2h at room temperature, they subsequently got exposed to primary antibodies at 4°C for a whole night. After having the membranes washed, they were sent for incubation with appropriate secondary antibodies, and subjected to detection by ECL Western blotting detection system. The following primary antibodies were used: anti-Nrf2 (1:2000, Proteintech), anti-HO-1 (1:2000, Hua-bio), anti-iNOS (1:1000, Affinity), anti-CD86 (1:1000, Affinity), anti-IL-10 (1: 2000, ABclonal), anti-Arg-1 (1:1000, Affinity) and anti-β-actin (1:2000, Proteintech).
3
Western Blot Analysis of Inflammatory Cytokines
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The protein concentrations were determined using an enhanced BCA protein assay kit (Proteintech, China). Samples containing equivalent amounts of protein (30 μg) were subjected to 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by isolation and transfer onto polyvinylidene fluoride membranes (PVDF, Millipore, USA). After blocking, the membranes were incubated with primary antibodies overnight at 4 °C. After washing with tris-buffered saline-Tween 20, the membranes were incubated with a secondary antibody for 1 h. Enhanced chemiluminescence (BioVision) was used for protein detection, and the reactive bands were visualized using ChemStudio Imaging (Analytikjena, German). The intensities of specific bands were quantified using ImageJ software, and β-actin was used as a loading control. The primary antibodies used: anti-interleukin [IL]-1β (1:1000, Abcam), anti-IL-10 (1:1000, Abcam), anti-TNF-α (1:1000, Santa Cruz), and anti-β-actin (1:5000, Abcam).
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