Protein was extracted from treated tissues by the whole protein extraction kit (WLA019, Wanleibio, Shenyang, China) and the nuclear protein and plasma protein kit (WLA020, Wanleibio) according to the manufacturer's instructions, respectively. In brief, ankle joint tissues were lysed in lysis containing 1% phenylmethylsulfonyl fluoride on ice for 5 min. After centrifugation (4°C, 12000 rpm, 10 min), the supernatant was collected as the total protein. The concentration of the protein was determined by the BCA kit (WLA004, Wanleibio). Proteins were electrophoresed on 5%, 8%, 10%, and 15% SDS-PAGE and then electrotransferred to PVDF membranes (IPVH00010, Millipore, USA). The membranes were blocked with 5% skimmed milk in TBST for 1 h and incubated with primary antibodies overnight at 4°C, respectively. After being washed with TBST, the membranes were incubated with the secondary antibody (37°C, 45 min). The blots were detected using the ECL kit (WLA003, Wanleibio) and quantified with the Gel-Pro-Analyzer system (WD-9413B, Liuyi, China). β-Actin and histone H3 were utilized as internal references, respectively. The antibodies used in this study were diluted with 5% skimmed milk, and the information is listed in Table 2 .
Bca kit
Manufactured by Wanlei
Sourced in China
The BCA kit is a colorimetric assay used for the quantitative determination of total protein concentration. It utilizes the bicinchoninic acid (BCA) method to measure the reduction of Cu2+ to Cu+ by protein in an alkaline medium, with the purple-colored reaction product being measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
β actin, by Wanlei (2 mentions)
Wla019, by Wanlei (1 mentions)
Ecl kit, by Wanlei (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Runx2, by Wanlei (1 mentions)
Col1a1, by Wanlei (1 mentions)
Goat anti rabbit igg hrp, by Wanlei (1 mentions)
Slc27a3, by Proteintech (1 mentions)
Stau1, by Affinity Biosciences (1 mentions)
Protein extraction kit, by Wanlei (1 mentions)
β actin, by Huabio (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
7 protocols using bca kit
1
Protein Extraction and Western Blot Analysis
2
Osteogenic Protein Expression Analysis
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On day 14 of osteogenic induction, lysis solution was added to the OM and PRP groups to extract protein, and protein quantification was performed using a BCA kit (Wanleibio, China). Proteins were isolated by electrophoresis using an SDS–PAGE gel kit (Wanleibio, China) and then transferred to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% skimmed milk powder (Yili, China) for 1 h and then incubated with primary antibody. The following primary antibodies were used: alkaline phosphatase (ALP) (Abclonal, China), Runx2 (Wanleibio, China), OCN (Affinity, China), COL1A1 (Wanleibio, China), CNR1 (Abclonal, China), and β-actin (Wanleibio, China). After four washes (5 min each), the membrane was then incubated with secondary antibody. The membrane was washed six times with Western detergent (Wanleibio, China), and enhanced chemiluminescence (ECL) solution (Wanleibio, China) was applied for exposure in a dark room. The film was scanned, and the optical density of the target protein was analyzed with Gel-Pro Analyser software.
3
Protein Expression Analysis by Western Blot
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Total proteins were extracted using RIPA lysis buffer. Total proteins were quantified using BCA kit (Wanleibio, Shenyang, China). Proteins were separated by SDS-PAGE, and were transferred onto PVDF membrane (Millipore, Billerica, MA, US). The membrane was blocked in defatted milk powder, and was incubated at 4 °C overnight in primary antibody (mouse anti-E-cadherin at 1:500; rabbit anti-α-SMA at 1:500; goat anti-Snail 1:400 and goat anti-MGAT3 1:200; Wanleibio, Shenyang, China). On the following day, the membrane was rinsed in PBS, and was incubated in secondary antibody (goat anti-mouse IgG-HRP 1:5000; goat anti-rabbit IgG-HRP 1:5000; donkey anti-goat IgG-HRP 1:5000; Wanleibio, Shenyang, China) for 1 h at room temperature. ECL chromogenic substrate (BD Bioscience, US) was added for development, and was imaged using a gel imaging system. Relative protein expression was quantified against the beta-actin band.
4
Protein Expression Analysis in Lung Tissues
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Total protein was extracted from lung tissues and BEAS-2B cells by protein extraction kit (Wanleibio). The concentration of protein was detected by BCA kit (Wanleibio). The protein was separated by SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane was blocked by non-fat milk and then incubated with primary antibodies for SLC27A3 (Proteintech), STAU1 (Affinity), ACPL2 (Bioss), RABL4 (Biorbyt), and β-actin (Wanleibio), respectively. After 12-h of incubation at 4°C, the membrane was washed, incubated by the secondary antibody, and visualized by CEL (Wanleibio). β-actin serves as an internal control.
5
Protein Quantification and Western Blot Analysis
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After 48 hours, the treated G401 and WT-CLS1 cells were washed by pre-cooled PBS for 3 times, lysed and centrifuged at 12000 rpm at 4°C for 10 minutes. After that, the supernatant was centrifuged in a 0.5 mL centrifuge tube and stored at −20°C. Next, protein quantification was performed through bicinchoninic acid (BCA) kit (Wanlei Bio, Shenyang, China). Then, the protein was added with 6×SDS loading buffer at 100°C for protein denaturation, separated by SDS electrophoresis, and transferred into membranes for 1.5 hours. After being blocked with the 5% skimmed milk powder configured with Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 hour, the membrane was incubated with primary antibody of CRK (ab45136, 1:1000) or β-actin (ab4970 s, 1:1000) overnight at 4°C. All the primary antibodies were purchased from Abcam (Cambridge, UK). The membrane was re-probed with goat anti-rabbit IgG or goat anti-mouse IgG at room temperature (1:5000, Beijing ComWin Biosciences Co., Ltd., China) for 2 hours. After TBST washing, electrochemiluminescent substrate A and B (Thermo Scientific Pierce Company, USA) were mixed in a ratio of 1:1 and then dropped on the membrane, followed by color development and exposure. Using β-actin as an internal reference, ImageJ was used to calculate the gray value of each band, and 3 replicate experiments were set for each cell line.
6
Protein Expression Analysis of VSMCs
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Protein of VSMCs was extracted with cold RIPA buffer (Beyotime, Shanghai, China) and measured using a BCA kit (Wanleibio, Shenyang, China). Protein lysates were then separated with SDS-PAGE and transferred to PVDF membranes. After blocked with 5% skimmed milk, membranes were incubated with primary antibodies against runt-related transcription factor 2 (Runx2) (1:1000; CST, USA), alpha smooth muscle actin (α-SMA) (1:2000; Huabio, China) and β-actin (1:2000; Huabio, China) at 4 °C overnight. After washed with TBST buffer, membranes were incubated with HRP-conjugated IgG secondary antibody (1:1000; Beyotime, China) for 1 h at room temperature. The Pierce ECL Western Blotting Substrate (Thermo, USA) was used for visualization. Quantization of bands was performed using the Image J software.
7
Quantifying Calcium Content in Vascular Smooth Muscle Cells
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Ten days after culture, Alizarin red S staining and quantification of cell calcium content were performed. For Alizarin red S staining, cultured VSMCs were fixed with 4% paraformaldehyde solution for 20 min at room temperature, washed with PBS three times, and stained with 1% Alizarin Red S solution (pH = 4.2) for 30 min at 37 °C. After staining, samples were washed with PBS and then observed under an inverted microscope. To quantify Ca2+ content of the cultured VSMCs, ultrasonic decomposition was performed after adding deionized water; next, supernatant was collected after centrifugation. Detection reagents from a commercial calcium assay kit (Nanjing Jiancheng Biotechnology Institute, Nanjing, China) were then added to supernatant following the manufacturer’s instructions. The OD value (detected at 610 nm) and protein concentration of samples measured using a BCA kit (Wanleibio, Shenyang, China) were used to determine the cell calcium content.
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