Total protein was extracted from brain tissue collected from the ischemic cortical region of rats using the conventional total protein extraction method. Rat brain tissues (n = 4), which had been preserved before retrieval, were extracted with lysis solution (Servicebio, Wuhan, China) for total protein, and the protein concentration was determined by the BCA protein quantitative assay kit (Servicebio, Wuhan, China). Samples were denatured in boiling water, and proteins were separated by electrophoresis and transferred to a methanol activated PVDF membrane (0.45 μm). Nonfat milk was added and blocked for 30 min at room temperature; with antibody to AMPK β 1 (Abclonal, A12491, 1:1,000), P-AMPK β 1 (Abclonal, A12491, 1:1,000), TFAM (Abclonal, A13552, 1:1,000), PGC1 α (Abclonal, A12348, 1:1,000), NRF2 (Abclonal, A0674, 1:1,000), UCP2 (Servicebio, GB11377, 1:1,000) were incubated overnight at 4°C, followed by the addition of secondary antibody reaction for 30 min at room temperature. The protein bands were chemiluminescent with the addition of mixed ECL solution, followed by development with developing, developing reagents, and processing with Photoshop and Alpha software to analyze the densitometric values of the target bands.
Bca protein quantitative assay kit
Manufactured by Wuhan Servicebio Technology
The BCA protein quantitative assay kit is a laboratory reagent used for the determination of protein concentration. It utilizes the bicinchoninic acid (BCA) method, which is a colorimetric detection and quantification technique. The kit provides the necessary solutions and reagents to perform this assay.
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Lab products found in correlation
2 protocols using bca protein quantitative assay kit
1
Protein Expression in Ischemic Rat Brains
2
Western Blot Analysis of Hedgehog Pathway
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Eca109 and Eca109R cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology) for 30 min at room temperature. Cells were centrifuged at 12,000 × g for 15 min at 4°C and the supernatant was collected. The BCA Protein Quantitative Assay kit (Servicebio) was used to determine the protein concentration. The samples were denatured by boiling prior to separation (35 µg per lane) using 10% SDS-PAGE followed by transfer to nitrocellulose membranes. Skimmed milk (5%) was used as a blocking agent for 1.5 h at room temperature. Next, the membranes were incubated on a shaker with the primary antibodies against Gli1 (1:2,000; cat. no. NBP2-45872; Novus Biologicals), Shh (1:5,000; cat. no. ab53281; Abcam), Smo (1:200; cat. no. GTX60154; GeneTex, Inc.), Ptch (1:500; cat. no. GTX83771; GeneTex, Inc.) and β-actin (1:5,000; cat. no. GTX629630; GeneTex, Inc.), overnight at 4°C, washed with Tris-buffered saline with 0.05% Tween-20, and incubated with secondary antibodies (1:4,000; cat. nos. 1031-05 and 4050-05; Southern Biotech) for 1 h at 37°C. Bands were detected using enhanced chemiluminescence substrate (Bio-Rad Laboratories, Inc.), and grayscale images were analyzed with ImageJ 15.0 software (National Institutes of Health). This procedure was performed three times.
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