Serum D-lactic acid and DAO levels, as well as interleukin (IL )-1β, IL-8, IL-10, IL-17, tumor necrosis factor-α (TNF-α ), interferon-γ (IFN-γ ), and secretory immunoglobulin A (sIgA ) levels in ileal mucosal were measured using enzyme-linked immunosorbent assay kits (Meimian Industrial Co., Ltd., Yancheng, China). Protein concentration in ileum mucosa was measured using the enhanced BCA protein assay kit (Biyuntian Biotechnology Co., Ltd., Shanghai, China), and the results were expressed as per milligram of protein.
Enhanced bca protein assay kit
Manufactured by Beyotime
Sourced in China, United States, Japan, Germany
The Enhanced BCA Protein Assay Kit is a colorimetric assay used for the quantification of protein concentration. The kit utilizes bicinchoninic acid (BCA) to detect and quantify total protein in a sample. The color change produced in the reaction between protein and BCA is measured spectrophotometrically, allowing for the determination of protein concentration.
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Lab products found in correlation
Ripa lysis buffer, by Beyotime (112 mentions)
Pvdf membrane, by Merck Group (78 mentions)
Ripa buffer, by Beyotime (47 mentions)
Polyvinylidene fluoride membrane, by Merck Group (33 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (32 mentions)
Polyvinylidene difluoride membrane, by Merck Group (25 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (21 mentions)
Beyoecl plus, by Beyotime (17 mentions)
Cell lysis buffer, by Beyotime (16 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (12 mentions)
Lipid peroxidation mda assay kit, by Beyotime (11 mentions)
Bcl 2, by Abcam (10 mentions)
Lysis buffer, by Beyotime (9 mentions)
Atp assay kit, by Beyotime (9 mentions)
Ripa lysis buffer, by Solarbio (9 mentions)
Pvdf membrane, by Bio-Rad (9 mentions)
β actin, by Abcam (8 mentions)
Sds lysis buffer, by Beyotime (8 mentions)
Protease inhibitor cocktail, by Roche (7 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (7 mentions)
672 protocols using enhanced bca protein assay kit
1
Gut Immune Biomarkers Analysis
2
Liver Protein Extraction and Western Blot Analysis
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Liver protein was extracted using a commercial protein extraction kit (Thermo Pierce, NO. 78510). Whole protein was quantified with an Enhanced BCA Protein Assay Kit (Biyuntian, China). The liver proteins (60 μg per lane) were probed with anti-LBP antibody (Abcam, No. ab25094), anti-CD14 antibody (Abcam, No. ab182032) and β-Actin antibody (Santa Cruz, No. SC-47778). Three volumes of protein solution were combined with one volume loading buffer. Sixty micrograms of proteins were loaded onto 10% SDS–PAGE gels. Electrophoresis was performed at 60 V for 2 h at 4°C. Then the proteins were blotted by electrodiffusion for 2 h at 100 V on nitrocellulose membranes. The membranes were blocked with 5% skim milk in T-TBS for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibody in T-TBS containing 5% skimmed milk powder. Then the membranes were rinsed in T-TBS for 5 min and repeated for four times. After that, the membranes were incubated for 1 h with goat anti-rabbit IgG (H+L) (Thermo Pierce, No. 31160) or goat anti-mouse IgG (H+L) (Thermo Pierce, No. 31210) secondary antibodies, and rinsed in T-TBS for 5 min and repeated five times. Finally, blots were detected using SuperSignal® West Dura Extended Duration Substrate according to the manufacture's protocol. The western blot images were analyzed by using the Quantity One software (Biorad).
3
Purification of His-tagged Proteins
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E. coli BL21(DE3)-LCC ICCG was grown at 37 ℃ in LB medium with appropriate antibiotics to an optical density of 0.8 at 600 nm. IPTG was added at a concentration of 0.2 mM to induce the expression. After cultivation for another 20 h at 37 ℃, culture supernatant was harvested by centrifugation at 12,000 rpm for 30 min. The his-tagged proteins in the supernatant were purified using Ni-NTA Agarose (Qiagen, Hilden, Germany) by gravity-flow Ni-affinity chromatography. The NTA-Ni agarose column was equilibrated with the binding buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl), non-target proteins were washed off with the washing buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole), target proteins were eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 250 mM imidazole). The protein concentration was determined by Enhanced BCA Protein Assay kit (Biyuntian, Beijing, China) according to the instructions.
4
Quantification of DNMT Expression
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Protein extracts were prepared from uninfected, E6-, E7- and control shRNA-infected SiHa and CaSki cells at 48 and 96 h after infection as described previously [60 (link), 61 (link)]. Total protein concentration was measured with an Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu ). For electrophoresis, approximately 50 μg of protein per lane was resolved on 10% SDS-polyacrylamide gels and transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA ). Membranes were incubated with a rabbit monoclonal antibodies against human DNMT1 and DNMT3A protein at 1:1000 dilution (CST, Boston, MA ); human DNMT3B and DNMT3L at 1:2000 dilution (Abcam, Cambridge, UK ); β-actin (CST, 1:1000 dilution) was used as internal control for protein loading and analysis. Bound antibody was detected by 20 × LumiGLO® Reagent and 20 × Peroxide (CST).
5
Purification and Characterization of Leuk-1 Cell-Derived Extracellular Vesicles
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EVs derived from Leuk-1 cells (Leuk-1-EVs) were purified by sequential ultracentrifugation (17 (link)) (Figure 1A
The ultrastructure and size of EVs were analyzed by transmission electron microscopy (TEM) (Hitachi, HT7800/HT7700, Japan) and subjected to nanoparticle tracking analysis (NTA) with a Zeta View® system (Particle Metrix, Germany). The protein concentration was measured by BCA assay (Enhanced BCA Protein Assay Kit, Beyotime Institute Biotechnology, China). Western blotting was performed to detect the protein markers of EVs, including CD63 (ab, 59479, Abcam, Cambridge, UK), TSG101 (ab 83, Abcam, Cambridge, UK), and HSP70 (10995-1-AP, Proteintech, Wuhan, China).
The ultrastructure and size of EVs were analyzed by transmission electron microscopy (TEM) (Hitachi, HT7800/HT7700, Japan) and subjected to nanoparticle tracking analysis (NTA) with a Zeta View® system (Particle Metrix, Germany). The protein concentration was measured by BCA assay (Enhanced BCA Protein Assay Kit, Beyotime Institute Biotechnology, China). Western blotting was performed to detect the protein markers of EVs, including CD63 (ab, 59479, Abcam, Cambridge, UK), TSG101 (ab 83, Abcam, Cambridge, UK), and HSP70 (10995-1-AP, Proteintech, Wuhan, China).
6
Protein Extraction and Western Blot Analysis
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Cells were lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China), and the protein concentration was determined using an enhanced BCA protein assay kit (Beyotime Biotechnology). Ten micrograms of each protein sample were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes. Samples were then incubated with specific primary antibodies overnight, following incubation with an antimouse secondary antibody solution for 2 h at room temperature. The blotted membranes were exposed to film, and the protein bands were visualized by a scanner system.
7
Purification of SARS-CoV-2 Nsp13 Helicase
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pET-28a-nsp13 and pET-28a-nsp13-K289A were transformed into Transetta (DE3) cells (TransGen Biotech, China), grown at 37℃, and induced with IPTG (0.2 mM) when the optical density reached ∼0.8. Thereafter, the induced cells were grown at 18℃ for 16 h. Cells were harvested at 15,000×g by centrifugation at 4℃. After ultrahigh-pressure disruption and centrifugation at 20,000×g, the supernatants were filtered through a 0.45-μM filter (Millipore, MA, USA), then run through a Ni-affinity column. The proteins were then eluted with a linear-gradient concentration of imidazole from 20 to 250 mM. The eluates were concentrated, and the buffers were replaced with Buffer B2 (0.2 M Tris−HCl, 200 mM NaCl) using Amicon Ultra-15 filters (Millipore). The concentration of purified protein was determined using an enhanced BCA protein assay kit (Beyotime, China).
8
Biochemical Assays for Kidney Injury
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Serum creatinine (Scr) and blood urea nitrogen (BUN) assay kits were purchased from Nanjing Jiancheng Biotech Co., Ltd. (Nanjing, Jiangsu, China). Enzyme-linked immunosorbent assay (ELISA) kit to determine the concentration of urinary albumin (UAlb) was purchased from JinYiBai Biological Technology Co., Ltd. (Nanjing, Jiangsu, China). Enhanced BCA Protein Assay Kit was obtained from Beyotime Institute of Biotechnology (Nanjing, Jiangsu, China). Anti-α-smooth muscle actin (α-SMA) antibody (ab32575) was purchased from Abcam (Cambridge, UK). Anti-vimentin (5741) antibody and anti-eNOS antibody (32,027) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-collagen I (AF7001) was obtained from Affbiotech (Cincinnati, OH, USA). Anti-GAPDH (MB001) was purchased from Bioworld Technology (St. Louis Park, MN, USA). The IRDye (680RD or 800CW)-labeled secondary antibodies were purchased from LI-COR Biotechnology (Lincoln, NE, USA). ChamQ SYBR qPCR Master Mix (Low ROX Premixed) (Q331-02) and RNA isolater Total RNA Extraction Reagent (R401-01) were purchased from Vazyme Biotech Co., Ltd. (Nanjing, Jiangsu, China). All the inorganic chemicals were from Sigma-Aldrich (St. Louis, MO, USA).
9
Western Blot Analysis of Apoptosis and PI3K/Akt Signaling
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NPMSCs were collected and lysed in lysis buffer (Beyotime, Jiangsu, China) containing a mixture of protease inhibitors phenylmethanesulfonyl fluoride (PMSF, Beyotime) and phosphatase inhibitor cocktail I (Sigma, USA). Protein concentrations in cell lysates were determined using an enhanced BCA protein assay kit (Beyotime). Whole lysates were separated using SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Amersham Biosciences, USA). Membranes were blocked with 5% bovine serum albumin (BSA, Beyotime) in Tris-buffered saline and Tween 20 (TBST) for 1 h at room temperature. Membranes were then incubated overnight at 4°C with the primary antibodies against Bax (1 : 1000, Abcam, UK), caspase-3 (1 : 1000, Abcam, UK), Bcl-2 (1 : 1000, Abcam, UK), p-Akt (phospho-S473, 1 : 1000, Abcam, UK), Akt (1 : 1000, Abcam, UK), PI3K (1 : 1000, Abcam, UK), p-PI3K (phospho-Tyr524, 1 : 1000, Thermo Fisher, US), and β-actin (1 : 5000, Abcam, UK). Membranes were washed three times with 0.1% Tween 20 in TBS for 10 min and incubated with the respective peroxidase-conjugated secondary antibodies for 2 h. After three 10 min washes, the proteins were visualized using an enhanced chemiluminescence (ECL) method according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, USA).
10
Western Blot Analysis of Apoptosis and Nrf2 Pathway
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Proteins were extracted from cell lysates, using RIPA lysis buffer, and the protein concentration was measured using the Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology), following the manufacturer’s recommendations. Proteins were denatured by heat, and then separated by SDS-PAGE electrophoresis, and finally transferred to PVDF membranes. The membranes were blocked using 1% bovine serum albumin solution for 1 h at room temperature, and then incubated with primary antibodies, including anti-cleaved caspase-3 (1:1000; Abcam), anti-Keap1 (1:1000; Abcam), anti-Nrf2 (1:1000; Abcam), anti-p-Nrf2 (1:1000; Abcam), anti-heme oxygenase-1 (1:1000; Abcam), anti-NAD(P)H quinone dehydrogenase 1 (1:1000; Abcam) and anti-β-actin (1:1000; Zhongshan Jinqiao Biotechnology, Beijing, China) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibody (1:5000; Zhongshan Jinqiao Biotechnology, Beijing, China) at room temperature for 30 min. Protein bands were detected using a Immun-Star HRP kit (Bio-Rad), following the manufacturer’s protocols. Relative densitometry was analyzed using the Image J2x analysis software (National Institutes of Health, USA).
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