The reaction mixture included 0.1 ml of purified enzyme solution and 0.9 ml of 0.3% sodium alginate (w/v) in PB containing 100 mM NaCl (pH 7.3) and was incubated for 10 min at 30 °C. The alginate lyase activities of recombinant VxAly7D and the mutants were assayed by measuring the increase in the absorbance at 235 nm (A235) for the unsaturated product. One unit (U) of activity was defined as an increase in the A235 value of 0.1 per min. The protein concentration was measured with BCA Protein Quantification Kits (Vazyme Biotech Co. Ltd., Nanjing, China).
Bca protein quantification kit
Manufactured by Vazyme
Sourced in China, United States
The BCA Protein Quantification Kit is a reagent-based solution for the quantification of protein concentrations. It utilizes the bicinchoninic acid (BCA) assay principle to measure the total protein content in a sample.
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Lab products found in correlation
Ripa buffer, by Beyotime (10 mentions)
Pvdf membrane, by Merck Group (9 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Sds page, by Solarbio (5 mentions)
Sodium dodecyl sulfonate polyacrylamide gel, by Solarbio (5 mentions)
Ab9485, by Abcam (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Ab205719, by Abcam (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
Ecl reagent, by Vazyme (3 mentions)
Ripa lysis buffer, by Solarbio (3 mentions)
Pvdf membrane, by Roche (3 mentions)
Ab181602, by Abcam (3 mentions)
Bs 0295m hrp, by Bioss Antibodies (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Xmark, by Bio-Rad (2 mentions)
Cleaved caspase 3, by Santa Cruz Biotechnology (2 mentions)
Ecl kit, by Vazyme (2 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (2 mentions)
β actin, by Sino Biological (2 mentions)
87 protocols using bca protein quantification kit
1
Alginate Lyase Activity Assay
2
Quantification of Inflammatory Mediators
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Cells were cultured and treated as previously described in a 6-well plate. Cell protein was extracted using Total Protein Extraction Kits and quantified using BCA Protein Quantification Kits (Vazyme, China). The contents of TNF-α, IL-1β, IL-6, COX-2, and iNOS were assessed using commercial ELISA kits (Sinobestbio, China) according to the manufacturer’s instructions. Each ELISA reaction was performed in triplicate. The absorbance values were measured at 450 nm in xMarkTM (Bio-Rad).
3
Western Blot Analysis of Signaling Pathways
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Cells were cultured and treated as previously described in a 6-well plate. Cell protein was extracted using Total Protein Extraction Kits, and quantified using BCA Protein Quantification Kits (Vazyme, China). For each sample, 20 μg protein was separated in 12% SDS-PAGEs. The protein bands were transferred to a polyvinylidene fluoride (PVDF) membrane and then blocked in Tris-buffered saline containing 0.05% Tween 20 and 5% bovine serum albumin for 2 h. The PVDF membranes were incubated at 4°C overnight with specific primary antibodies (1:1000 dilution in TBST) against p65, p-p65, IκBα, p-IκBα, p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2. On the next day, the PVDF membranes were washed with TBST 5 times for 8 min each, and incubated with Rabbit Anti-Goat IgG/HRP (Bioss, China) antibody in 1:1000 dilution in TBST for 1 h at room temperature. After washing with TBST as described above, the protein bands were visualized with ECL western blotting detection reagent (Bioground, China). The experiments were repeated three times. β-actin protein was used as an endogenous control as in the previous report in MAC-T cells [20 (link)]. Band intensity normalized to β-actin was evaluated using Image Lab software (version 5.2.1, Bio-Rad).
4
Immunofluorescence and ELISA Analysis of NF-κB p65 Translocation
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For immunofluorescence, MAC-T cells were cultured in 96-well plates until 5,000 cells per well, and they were treated as previously described. The cells were incubated with the primary antibody against p65, followed by incubation with Rabbit Anti-Goat IgG/Cy3 antibody (Bioss). The nuclei were stained blue with 4’, 6-diamidino-2-phenylindole (DAPI) and observed with an inverted fluorescent microscope (Leica, Germany).
For an ELISA test of p65 nuclear translocation, cells were cultured and treated as previously described in a 6-well plate. Nuclear protein was extracted using Nuclear Protein Extraction Kits, and was quantified using BCA Protein Quantification Kits (Vazyme, China). The content of NF-κB p65 in the nucleus was assessed using commercial ELISA kits as previously described.
For an ELISA test of p65 nuclear translocation, cells were cultured and treated as previously described in a 6-well plate. Nuclear protein was extracted using Nuclear Protein Extraction Kits, and was quantified using BCA Protein Quantification Kits (Vazyme, China). The content of NF-κB p65 in the nucleus was assessed using commercial ELISA kits as previously described.
5
Quantification of Inflammatory Markers
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Cells were seeded in a 6-well plate and a 24-well plate for the detection of cytokines and endogenous endotoxin, respectively. Cell protein was extracted and quantified with Total Protein Extraction Kits and then BCA Protein Quantification Kits (Vazyme, Nanjing, China). The contents of IL-1β, TNF-α, IL-6, iNOS and COX-2 were detected by commercial ELISA kits (Youxuan, Shanghai, China) in triplicate, while endogenous endotoxin in cells was determined using Endotoxin (ET) ELISA kits (Jiangsu Jingmei Biological Technology, Yangcheng, China ). The OD values at 450 nm were measured in xMarkTM (BIO-RAD, California, America).
6
DBP and MWCNT Exposure Evaluation
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Reagents: DBP was purchased from Sigma-Aldrich, USA. MWCNTs (outer diameter less than 8 nm, length 0.5–2 μm) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China, File S1 ).
Kits: The DBP ELISA kit was purchased from Prime Bio Tek. BCA Protein Quantification Kit was purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). GSH detection kits were purchased from Nanjing Jiancheng Bioengineering Institute.
Kits: The DBP ELISA kit was purchased from Prime Bio Tek. BCA Protein Quantification Kit was purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). GSH detection kits were purchased from Nanjing Jiancheng Bioengineering Institute.
7
Western Blot Protein Quantification and Detection
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Proteins were extracted by using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) and then quantified using a BCA Protein Quantification Kit (#E112-01, Vazyme, China). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.45 μm polyvinylidene fluoride (Millipore, USA). The membrane was blocked using 5% nonfat dry milk in Tris‐buffered saline solution containing 0.1% Tween 20 (TBS‐T) for 1 h at room temperature and then probed with specific primary antibodies overnight at 4 °C. Subsequently, The membranes were washed with TBS‐T, followed by incubating with HRP-conjugated secondary antibodies for 1 h at room temperature. β-actin was used as the protein loading control. Protein signals were developed with SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, USA) and imaged using a chemiluminescence imaging system MiniChemi™ 610 with SageCapture TM software version 2.17.12.170316 (Sagecreation, China).
8
Protein Expression Analysis by Western Blot
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Cells were harvested and homogenized in RIPA lysis buffer and centrifuged at 12,000 rpm for 20 min at 4°C. Protein concentrations of the supernatants were determined using BCA Protein Quantification Kit (Vazyme biotech co., Itd.). Western blot analysis was performed as previously described [38 (link)], using rabbit polyclonal antibodies to human survivin and c-IAP1 (R&D systems, MN), XIAP and Cyclin B1 (Cell signaling, MA), phosphor-Cdc25C, Cdc2, phospho-Cdc2, cleaved PARP and cleaved caspase-3 (Santa Cruz, CA).
9
Protein Extraction and Western Blot Analysis
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The RIPA buffer was used to extract the total protein (Solarbio, Beijing, China). Protein quantification was done with the BCA Protein Quantification Kit (Vazyme). After that, following electrophoresis by SDS-PAGE, the separated proteins (50 µg) were electroblotted from the gel on PVDF membranes (Sigma-Aldrich). followed by an overnight treatment at 4 °C with primary antibodies: CDKN2C (ab192239, Abcam, Cambridge, USA), Cyclin D1 (60186-1-Ig, Proteintech, Wuhan, China), RB1 (ab181616, Abcam), p-RB1 (ab184796, Abcam), CDK4 (11026-1-AP, Proteintech), Bcl-2 (12789-1-AP, Proteintech), BAX (50599-2-Ig, Proteintech), and then 1.5-h interaction with secondary antibody (Abcam) at room temperature (RT). Chemiluminescence was seen with the ECL kit (Vazyme).
10
Western Blotting Protein Expression Analysis
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Western blotting was performed as described previously 36 (link). The protein samples were prepared by a total cell protein extraction kit (Vazyme, Nanjing, China). BCA protein quantification kit (Vazyme) was used for concentration determination. 40 μg protein samples were transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then blocked with fat-free milk. The primary antibodies against E2F1 (1:1000, #ab112580, Abcam, Cambridge, UK), β-actin (1:2000, #66009-1-Ig, ProteinTech, Chicago, IL, USA) were used. ECL kits (Beyotime Biotechnology, Beijing, China) and IMAGE J software (National Institutes of Health, Bethesda, MD, USA) were used to quantify the signal intensity.
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