Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, USA). Ursolic acid (UA) was purchased from Nanjing Zelang Plant Extract Co., Ltd. (Nanjing, China). Epirubicin was purchased from Rhawn (Shanghai, China). Autophagy inhibitor, 3-Methyladenine (3-MA), was purchased from Selleck Chemicals (Houston, TX, USA). Protease inhibitor, ECL luminescence reagent, Cell lysis buffer, Cell Counting Kit-8, Caspase3 ELISA Kit, Bradford Protein Assay Kit and autophagy agonist, rapamycin (RAPA), were obtained from Solarbio (Beijing, China). Class I PI3K, AKT, Beclin-1, LC3-II/LC3-, Atg5, Atg7 and β-Actin antibodies were purchased from CST (USA). RIPA Lysis Buffer (RIPA) was purchased from Biosharp (Guangzhou, China). Monodansylcadaverine (MDC) and Transwell chambers were purchased from Sigma (Shanghai, China). BCA Protein Assay Kit (BCA) was purchased from Bioteke (Beijing, China).
Bca protein assay kit
Manufactured by BioTeke
Sourced in China
The BCA protein assay kit is a colorimetric detection and quantification method used to measure the total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) reaction to produce a purple-colored complex, which can be measured spectrophotometrically. The core function of this kit is to provide a simple and accurate means of determining protein levels in various biological, chemical, or biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (3 mentions)
Quantity one 4, by Bio-Rad (2 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (2 mentions)
Bovine pancreatic rnase a, by Merck Group (2 mentions)
Rnasealert kit, by Integrated DNA Technologies (2 mentions)
Lysis buffer, by BioTeke (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Monodansylcadaverine mdc, by Merck Group (1 mentions)
Transwell chamber, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by Solarbio (1 mentions)
Ecl luminescence reagent, by Solarbio (1 mentions)
Caspase3 elisa kit, by Solarbio (1 mentions)
Rapamycin rapa, by Solarbio (1 mentions)
3 methyladenine 3 ma, by Selleck Chemicals (1 mentions)
Cell counting kit 8 (cck8), by Solarbio (1 mentions)
Bradford protein assay kit, by Solarbio (1 mentions)
Mmp14, by Abcam (1 mentions)
Mab 4060, by Cell Signaling Technology (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Vazyme (1 mentions)
17 protocols using bca protein assay kit
1
Ursolic Acid Inhibits Autophagy in Cells
2
Western Blot Analysis of ER Stress and MMP14
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The concentration of each sample protein was measured by the bicinchoninic acid (BCA) protein assay kit (Bioteke, Beijing, China). Equal amounts of total protein (50 μg) from each sample were separated in a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel for 3 h at room temperature and transferred to a polyvinylidene fluoride (PVDF) membrane at 110 V for 2 h on ice. The blot was blocked with 5% nonfat dry milk suspended in 1× Tris buffered saline with Tween (TBST) (25 mM Tris, 2.7 mM KCl, and 137 mM NaCl, 0.05% Tween-20) for 40 min in the incubator at 37 °C. Then, the blot was incubated with ER stress antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA) and MMP14 (1:1000, Abcam) at 4 °C for 12 h, followed by incubation with goat anti-rabbit IgG-HRP (1:10,000, Lianke, Hangzhou, China) for 1 h at room temperature. The blot was washed three times with 1× TBST for 10 min between the first and second incubation. A densitometer (Bio-Rad) to scan the bands and a Quantity One 4.6.3 software (Bio-Rad) was used for quantification. This experiment was repeated four times.
3
Evaluating PTEN/AKT Signaling in Embryonic Stem Cells
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Following appropriate periods of cultivation, ESCs were washed twice with PBS and then scraped into a lysate buffer containing 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml leupeptin, 2 mg/ml aprotinin, and 5 mM EGTA in PBS. The cells were sonicated using a sonifier cell disrupter and the resulting sonicates were centrifuged at 10,000 × g for 10 min. The supernatants were denatured in sample buffer and heated by boiling water for 5 min. The protein quantity was determined using a BCA protein assay kit (BioTeke Corporation, PP1001, China). Subsequently, the proteins were separated via 10% SDS-PAGE and electrophoretically transferred from the gels onto polyvinylidene difluoride (PVDF) transfer membranes. The membranes were blocked with 5% nonfat milk for 2 h, washed briefly in PBS-Tween, and then incubated overnight at 4 °C with primary antibodies against PTEN (1:1000, Cell Signaling, mAb #9559, USA), AKT (1:1000, Cell Signaling, mAb#4691, USA) and p-AKT (1:1000, Cell Signaling, mAb #4060, USA). An appropriate secondary antibody was applied for an additional 2-h incubation period, followed by protein expression evaluation using the Bio-Rad Imaging System (Bio-Rad Biosciences, USA), with GAPDH (1:1000, Cell Signaling, mAb #5174, USA) serving as an internal control for protein loading.
4
Cytotoxicity and Apoptosis Evaluation
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Branched PEI25K (impurities: ≤1% water) and bovine pancreatic RNase A (≥70 kU/mg protein) were purchased from Sigma-Aldrich (St. Louis, MS, USA). Genipin (>98%) was provided by Zhixin Biotechnol. Co. (Linchuan, China). RNaseAlert® kit was obtained from Integrated DNA Technologies, Inc. (Coralville, IA, USA). FBS and DMEM were purchased from Kangyuan Co. (Beijing, China) and Gibco (Grand Island, NE, USA), respectively. BCA protein assay kit was provided by BioTeke Co. (Beijing, China). Blue plus II protein marker was purchased from TransGen Biotech. (Beijing, China). BSA and MTT were purchased from Amresco (Solon, OH, USA). LIVE/DEAD® Viability/Cytotoxicity kit and one-step TUNEL cell apoptosis detection kit were obtained by Thermo Fisher (Grand Island, NE, USA) and Beyotime (Jiangsu, China), respectively. The Annexin V-FITC/PI apoptosis detection kit was provided by Vazyme Co. (Nanjing, China).
5
SDS-PAGE and Western Blot Analysis
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The protein concentration of each sample was measured using a bicinchoninic acid (BCA) protein assay kit (Bioteke, China). Equal amounts of total protein (50 μg) from each sample were separated in a 10% SDS-PAGE gel and transferred to a PVDF membrane at 120 V for 2 h at room temperature (RT). The blot was blocked with 5% nonfat dry milk suspended in 1x TBS (25 mM Tris, 137 mM NaCl, and 2.7 mM KCl) for 1 h at room temperature. Membranes were incubated with primary antibodies against STAT6 and GAPDH (Cell Signaling Technology, USA). The resulting blots were incubated with 1:1,000 rabbit–mouse secondary antibodies (Cell Signaling Technology, 4414/4410, USA). Bands were scanned using a densitometer (Bio-Rad) and quantified using the Quantity One 4.6.3 software (Bio-Rad).
6
PEEK Substrate Functionalization for Cell Culture
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PEEK (GEHR® PEEK-1000, Germany), sodium borohydride (NaBH4, Jinshan Chemical Reagent Co., LTD., Chengdu, China), dimethylsulfoxide (DMSO), 3-ammoniumpropyltriethoxysilane (APTES, Macklin, China), CPP (Solarbio, China), alpha-Minimum Essential Medium (α-MEM, Procell, China), fetal bovine serum (FBS, TIANHANG, China), 4% paraformaldehyde (Biosharp, China), 4′,6′-diamidino-2-phenylindole (DAPI, Solarbio, China), cell counting kit (CCK-8, Dojindo, Japan), actin-tracker green (Beyotime, China), NBT/BCIP ALP color development kit (Sangon Biotech, China), alkaline phosphate activity assay kit (Nanjing Jiancheng Biotechnology, China), bicinchoninic acid (BCA) protein assay kit (BioTeke, China), Alizarin Red S (ARS, Solarbio, China), RNAsimple Total RNA Kit (TIANGEN, China), ReverTra Ace® qPCR RT Master Mix (TOYOBO, Japan), SYBR Green Realtime PCR Master Mix (TOYOBO, Japan).
7
Protein Extraction and Western Blot
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Cells were harvested in a lysis buffer with a protease inhibitor cocktail (Roche). After assay of protein concentration using the BCA protein assay kit (Bioteke), equal amounts of protein were denatured, separated through SDS‐PAGE electrophoresis. Then, proteins were transferred onto polyvinylidene difluoride membranes. Afterward, the membranes were blocked, incubated with primary and secondary antibodies (Table S1 ), and finally visualized using a chemiluminescence imager (BIO‐RAD).
8
RNase A Cytotoxicity Assays
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Bovine pancreatic RNase A, zinc acetate, 2-methylimidazole, and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzymatic activity was detected by RNase Alert® kit, which was obtained from Integrated DNA Technologies, Inc. (Coralville, IA, USA). BCA protein assay kit was obtained from BioTeke (Beijing, China). The LIVE/DEAD® Viability/Cytotoxicity kit was provided by ThermoFisher (Grand Island, NE, USA). One-step TUNEL cell apoptosis detection kit (green fluorescence) and lactate dehydrogenase (LDH) cytotoxicity assay kit were purchased from Beyotime (Jiangsu, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Kangyuan Co. (Beijing, China) and Gibco (Grand Island, NE, USA), respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Amersco (Solon, OH, USA). All other reagents were of the highest grade available and used as received.
9
Adenoviral Gene Transfection Efficiency in HepG2 and 293T Cells
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In order to estimate the adenoviral gene transfection and expression efficiency in vitro, HepG2 and 293 T cells were infected with four recombinant adenoviruses at multiplicity of infection (MOI) of 40 and 4, respectively. After incubation for 48 h, the GFP expression and infection efficiency were determined under fluorescence microscopy. The cell protein and growth medium were collected for Western blot and ELISA analyses. The protein of the transfected HepG2 and 293 T cells were extracted by a protein extraction kit (Bio Teke Corporation, China), and the protein concentration was determined by the BCA Protein Assay Kit (Bio Teke Corporation, China), according to the manufacturer’s protocol. Thereafter, total protein (15 µg) was loaded into each lane of acrylamide gel and subjected to SDS-PAGE. The membranes were incubated with antibodies against human ApoE (ab183597, Abcam) at 4 °C overnight; α-tubulin antibody (sc-8035, Santa Cruz Biotechnology) was used as an internal control to verify the basal expression level and equal protein loading. Target protein bands were captured by the chemiluminescence imager (iBright CL1000, Thermo Scientific).
10
Chymase Modulation of TGF-β1 Signaling
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Cells were seeded into six-well plates at a density of 5×104 cells/well. The cells were incubated with various concentrations of chymase for 6, 12 and 24 h. Following incubation, the cells were washed with ice-cold phosphate-buffered saline, and lysed in 500 μl ice-cold radioimmunoprecipitation assay buffer (BioTeke Corporation, Beijing, China), containing 1 μg/ml phosphatase inhibitors and 1 mM phenylmethanesulfonyl fluoride, for 30 min. The mixture was centrifuged at 12,000 × g (4°C) for 10 min, after which the supernatants were stored at −80°C. The concentration of TGF-β1, phosphorylated Smad2/3 (P-Smad2/3), Smad2/3, Smad7 and GAPDH proteins were measured using a BCA protein assay kit (BioTeke Corporation).
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