Following the x-LDL treatment or transfection of Ov-Fibulin-5 plasmids plus ox-LDL treatment, or the co-treatment of transfection of Ov-YY1 and siRNA-Fibulin-5 plus ox-LDL treatment, VSMCs were collected and lysed by adding RIPA buffer (Beyotime Institute of Biotechnology) on ice for 30 min. The supernatant was collected after centrifugation at 4˚C at 14,000 x g for 20 min. The protein concentration of each group was detected by a BCA kit (Abcam). A total of 20 µg each protein sample was separated by 10% SDS-PAGE and then transferred into PVDF membrane. The membranes were then blocked with 5% BSA (MilliporeSigma) at room temperature for 2 h and incubated with primary antibodies (Fibulin-5; cat. no. ab109428, 1:1,000; cyclin D1; cat. no. ab16663, 1:200; CDK2; cat. no. ab32147, 1:1,000; MMP2; cat. no. ab92536, 1:1,000; MMP9; cat. no. ab76003, 1:1,000; GAPDH; cat. no. ab8245, 1:5,000. Abcam) at 4˚C overnight. After incubation with a secondary antibody at 37˚C for 2 h (Goat Anti-Rabbit IgG; cat. no. ab7090, 1:10,000. Abcam, England), a ECL color development reagent (cat. no. ab133409; Abcam) was added to the membranes. The density of the protein bands was analyzed using the ImageJ software (version 1.53b; National Institutes of Health).
Bca kit
Manufactured by Abcam
Sourced in United States, United Kingdom, China
The BCA kit is a protein quantification assay that uses bicinchoninic acid (BCA) to determine the total protein concentration in a sample. It is a colorimetric detection method that measures the reduction of copper ions by proteins in an alkaline environment, resulting in a purple-colored complex that can be detected spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Ripa buffer, by Beyotime (4 mentions)
Ab205718, by Abcam (4 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
Nitrocellulose membrane, by Solarbio (2 mentions)
Ripa buffer, by Solarbio (2 mentions)
Ab68153, by Abcam (2 mentions)
Ab254788, by Abcam (2 mentions)
Sc 47724, by Abcam (2 mentions)
Pvdf membrane, by Abcam (2 mentions)
Ecl reagent, by Beyotime (2 mentions)
Ab9485, by Abcam (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab3697, by Abcam (1 mentions)
Ab186838, by Abcam (1 mentions)
Ab227387, by Abcam (1 mentions)
Ecl reagent, by Solarbio (1 mentions)
Ab1031, by Merck Group (1 mentions)
High intensity ultrasonic processor, by Ningbo Scientz Biotechnology (1 mentions)
41 protocols using bca kit
1
Fibulin-5 and YY1 Regulation in VSMC
2
Western Blot Analysis of Chondrocyte Markers
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After lysing in RIPA buffer (Solarbio), cell lysates were centrifuged for protein collection. The concentration of protein sample was tested using a BCA kit (Abcam). Twenty microgram samples were loaded on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Solarbio). Next, the membranes were blocked in 5% non-fat milk, and then interacted with primary antibodies anti-Sry-type high-mobility-group box 9 (SOX9) (ab3697, 1:1000 dilution, Abcam), anti-collagen type II α 1 (COL2A1) (AB761, 1:100 dilution, Sigma–Aldrich), anti-Aggrecan (AB1031, 1:1000 dilution, Sigma–Aldrich), anti-TGFBR2 (ab186838, 1:500 dilution, Abcam) or anti-β-actin (ab227387, 1:5000 dilution, Abcam) and the secondary antibody conjugated by horseradish peroxidase (ab205718, 1:20000 dilution, Abcam). β-actin functioned as a loading control. Subsequently, the protein blots were developed via exposing to ECL reagent (Solarbio) and tested by ImageJ software (NIH, Bethesda, MD, U.S.A.). Relative protein level was normalized to the control group.
3
Wheat Proteome Extraction and Digestion
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Approximately 2 g tissues were collected from each frozen wheat sample, placed in a pre-cooled grinding tube, and ground into powders in liquid nitrogen. Each powdered sample was transferred into a 5 ml centrifuge tube, four volumes of a lysis buffer [8 M urea, 10 mM dithiothreitol, 1% Triton-100, 3 µM trichostatin A (TSA), 50 mM nicotinamide (NAM), and 1% protease inhibitor cocktail] were added into the tube, and then sonicated three times on ice using a high-intensity ultrasonic processor (Scientz, Ningbo, China). An equal volume of Tris-saturated phenol, pH 7.8, was added into the tube and the mixture was vortexed for 5 min followed by 15 min centrifugation at 10000 g and 4℃. The upper phenol phase was carefully transferred into a clean centrifuge tube. Four volumes of ammonium sulfate-saturated methanol was added into the tube and the tube was incubated at -20 ℃ for over 6 h to precipitate proteins. After 10 min centrifugation at 4 °C, the pellet was rinsed once with an ice-cold methanol and then three times with an ice-cold acetone. The resulting pellet was dissolved in an 8 M urea and its protein concentration was determined with a BCA kit (Abcam, Cambridge, MA, USA) as instructed. The trypsin was used for protein digestion, according to the description of Ye et al. [35 (link)].
4
Quantifying Scar-related Protein Changes
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Supernatants of TGFβ1-stimulated FBs after gene knockdown and treatment with protease inhibition were collected, centrifuged, and stored at −20 °C for further use. Protein levels of human procollagen Ia1 ELISA (R&D Systems) and human fibronectin ELISA (R&D Systems) were measured according to the manufacturer’s manual. Absorbance was detected by FluoStar Optima microplate reader (BMG Labtech, Ortenberg, Germany). Six-millimeter punch biopsies of healthy skin and hypertrophic scar tissue were lysed in 1% Triton X-100 lysis buffer (Sigma) and mechanically homogenized using precellyse tissue homogenizer. After centrifugation, lysates were analyzed using DPP4 and urokinase ELISAs (both R&D Systems) Total protein concentrations were measured using a BCA-kit (Abcam) according to the manufacturer’s protocol, and concentrations were normalized to total protein.
5
Western Blot Analysis of Histone Acetylation
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The treated cells were lysated with RIPA buffer (Abcam, Cambridge, UK) and collected for western blot. Protein quantification was performed using BCA kit (Abcam, Cambridge, UK). Protein (10 μg) was loaded for electrophoresis and transferred to PVDF membranes. The blots were blocked and labeled with primary antibodies (mouse anti-human HAT1, 1:2000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit anti-human KAT8, 1:2000 (Abcam, Cambridge, UK); or mouse anti-human β-actin 1:5000 (Santa Cruz Biotechnology, Dallas, TX, USA) overnight. Membranes were incubated with the secondary antibody (goat anti-rabbit-HRP or goat anti-mouse-HRP, 1:5000 dilution each; Amersham Pharmacia Biotech, Saclay, France). The signal was developed and imaged using ImageQuant™ LAS 4000 (Fujifilm, Tokyo, Japan).
6
Protein Extraction and Western Blot
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Total protein was extracted after lysis, and the protein concentration was detected using a BCA kit (Abcam, Cambridge, UK). The protein samples were denatured at 100 °C. After electrophoresis, membrane transfer, sealing, incubation with primary and secondary antibodies, and exposure, the proteins were visualized using photography. The MyoD antibody (ABclonal, Wuhan, China) and GAPDH antibody (Bioss, Beijing, China) were used for this assay.
7
SDS Lysis and Western Blot Analysis
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Tissues were lysed using SDS lysis buffer (containing 100 mmol/L Tris-HCl (pH = 7.0), 1% SDS and 2% 2-mercaptoethanol, supplemented with 1 x protease inhibitors from Roche) and incubated at 105 ℃ for 10 min. Total protein was extracted and quantified using the BCA Kit (Abcam). Equal amounts proteins were subjected to SDS-PAGE electrophoresis and subsequently transferred onto PVDF membranes (Millipore). After blocking with 5% skim milk, the membranes were treated overnight at 4 ℃ with the following antibodies: Mouse monoclonal antibody against GAPDH (1: 2000 dilution; 0411; sc47724); Rabbit monoclonal antibody against STAT3 (1: 1000 dilution; abcam; ab68153). Rabbit monoclonal antibody against STAT3 (1: 500 dilution; abcam; ab254788). Following incubation with HRP-conjugated secondary antibodies, the PVDF membrane was visualized using an enhanced chemiluminescence (ECL) kit (Thermo).
8
Western Blot Analysis of Protein Markers
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Total proteins from different groups were extracted with Trizol with protease inhibitors. Protein concentrations were determined using a BCA kit (Abcam, USA). Next, protein samples were separated by SDS-PAGE, and transferred onto PVDF membranes (BioRad, Hercules, CA). The membranes were incubated with the primary antibodies against GAPDH (1:1,000, Abcam), MMP2 (1:1,500, Abcam), MMP9 (1:1,000), and VEGF (1:1,000, Abcam) at 4°C for 12 h. After blocking in 5% nonfat milk for 1 h, the membranes were incubated with secondary antibodies at room temperature for 1 h and washed with Tris-buffered saline with Tween 20 (TBST). The signals were visualized using an enhanced chemiluminescence (ECL) reagent.
9
Limonin Modulates HK Activity in HepG2 Cells
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HepG2 cells were seeded into a 6-well plate and grown to 70%–80% confluence, and then the medium was replaced with fresh culture medium and incubated with different concentrations of limonin for 24 h. Cell numbers were counted, and cell pellets were obtained by centrifugation and then lysed with RIPA lysis buffer (cat 89900; Thermo Fisher Scientific). Protein concentration in cell lysate was determined by BCA Kit, and the HK activity in cell lysate was examined with the Hexokinase Assay Kit (cat ab136957; Abcam) by following the instructions of manufacturer. The HK activity in the group without limonin was calculated as 100%.
10
Protein Extraction and Western Blot Analysis
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Protein from cell cultures were extracted by freeze-and-thaw cycles. Muscles were cut into 40 slices, 20 μm thick and lysed with 100 μl of lysis buffer (50 mM Tris pH7.5, 150 mM NaCl, 10 mM MgCl2, 0.5 mM DTT, 1 mM EDTA, 10% glycerol, 2% SDS, 1% Triton from Sigma-Aldrich®, 1:50 Complete Phosphatase inhibitor from Roche®). The total protein concentration was determined using BCA kit (Abcam®, Prodotti Gianni, Milano, Italy). Cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and then western blotting. Primary antibodies were anti-p-AKT (Thr308, cat# 13038), anti-p-mTOR (Ser2448, cat# 2971), anti-p-S6 (Ser 235/236, cat# 4858), monoclonal anti-GAPDH (cat# 5174), anti-LC3 (cat# 4108), anti-P62 (cat# 8025, all from Cell Signaling®; dilution 1:1000), anti-Lys63 (cat# 05-1308), and anti-Lys48 (cat# 05-1307, both from Millipore®; dilution 1:500) (25 (link)). Immunoreactive bands were detected using proper HRP-secondary reagent and Clarity Western ECL Substrate (Bio-Rad).
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