Transfected cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) on ice for 15 min and centrifuged at 13,000 × g for 10 min at 4°C. Protein levels were measured by Enhanced bicinchoninic acid (BCA) Protein Assay Kit (Beyotime) and calculated evenly to load onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGEs) for the following blotting assays. Proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (Roche) using a semi-dry transfer cell (Bio-Rad, Hercules, CA, USA). After blocking with 5% skim milk, membranes were incubated with corresponding primary antibodies overnight at 4°C. Primary antibodies used in our study are obtained from Abcam (Cambridge, MA, USA).
Enhanced bicinchoninic acid bca protein assay kit
Manufactured by Beyotime
Sourced in China
The Enhanced Bicinchoninic Acid (BCA) Protein Assay Kit is a colorimetric assay for the quantitative determination of total protein concentration. The kit utilizes the well-established bicinchoninic acid (BCA) method for the detection and quantification of total protein.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (1 mentions)
Semi dry transfer cell, by Bio-Rad (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Roche (1 mentions)
Sirt1 sirna, by RiboBio (1 mentions)
Phenylmethanesulfonyl fluoride pmsf, by Beyotime (1 mentions)
Cell lysis buffer for western and ip, by Beyotime (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyl tetrazolium bromide mtt, by Roche (1 mentions)
Glutathione peroxidase gsh px detection kits, by Nanjing Jiancheng (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Lipofectamine 2000, by GenePharma (1 mentions)
Silymarin, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Gsh px, by Nanjing Jiancheng (1 mentions)
Tissue protein extraction kit, by Keygen Biotech (1 mentions)
Anti col1, by Abcam (1 mentions)
Anti col 3, by Abcam (1 mentions)
Anti α sma, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
7 protocols using enhanced bicinchoninic acid bca protein assay kit
1
Protein Extraction and Western Blotting
2
Biochemical Markers of Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Creatinine (Cr), blood urea nitrogen (BUN), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-Px) detection kits were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Enhanced Bicinchoninic Acid (BCA) Protein Assay Kit, Reactive Oxygen Species Assay Kit, Cell lysis buffer for Western and IP and phenylmethanesulfonyl fluoride (PMSF) were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Sodium dodecyl sulfate (SDS), hydroxymethyl aminomethane (Tris) and 4′,6′-Diamidino-2-phenylindole (DAPI) were purchased from Sigma (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was provided by Roche Diagnostics (Basel, Switzerland). Hematoxylin, 2-step plus®Poly-HRP Anti-Mouse/Rabbit IgG Detection System and 3,3′-Diaminobenzidine tetrahydrochloride (DAB) Substrate Kit were provided by Zhongshan Golden Bridge Biotechnology (Beijing, China). TransZol™, TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR(One-Step gDNA Removal) and TransStart® Top Green qPCR SuperMix were supplied by Beijing TransGen Biotech Co., Ltd. (Beijing, China). Sirt1 siRNA (Sequence: CCACCTGAGTTGGATGATA) was purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China), and Lipofectamine 2000 was obtained from GenePharma (Shanghai, China).
3
Hepatoprotective Effects of Natural Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). Silymarin was purchased from Sigma Chemical Company (Milan, Italy). Detection kits for ALT (Code No. C009-2), AST (Code No. C010-2), SOD (Code No. A001-1), MDA (Code No. A003-1), GSH-Px (Code No. A005), TG (Code No. A110-1), and TC (Code No. A111-1) were all supplied by Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). The Tissue Protein Extraction Kit was produced by KeyGEN Biotech. Co., Ltd. (Nanjing, China). The Enhanced Bicinchoninic Acid (BCA) Protein Assay Kit was purchased from Beyotime Institute of Biotechnology (Haimen, China). Tris (hydroxymethyl) aminomethane (Tris), sodium dodecyl sulfate (SDS), 4’,6’-Diamidino-2-phenylindole (DAPI), and Oil Red O were obtained from Sigma (St. Louis, MO, USA). Hematoxylin (Code No. ZLI9606), eosin (Code No. ZLI9612), and diaminobenzidine (DAB, Code No. ZLI9632) staining kits were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). TransZol, TransStart Top Green qPCR SuperMix, and Trans-Script All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) were purchased from Transgen Institute of Biotechnology (Beijing, China).
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fibroblasts were treated with lysis buffer. Total proteins of the cells were extracted using a Total Protein Extraction Kit (Keygen, Nanjing, China) supplemented with phenylmethylsulfonyl fluoride (PMSF), phosphatase inhibitors, and proteinase inhibitor. We measured protein concentration using an Enhanced Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China) and balanced them. After electrophoretic separation, transfer onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and blocking procedures, proteins were incubated overnight with anti-α-SMA (Abcam, Cambridge, MA, USA, 1: 1000), anti-Col-1 (Abcam, Cambridge, MA, USA, 1: 1000), anti-Col-3 (Abcam, Cambridge, MA, USA, 1: 1000), anti-fibronectin (Abcam, Cambridge, MA, USA, 1: 1000), and anti-GAPDH (Proteintech, Rosemont, IL, USA, 1: 10 000). After washing with Tris-buffered saline-tween (TBST), the proteins were incubated with secondary antibody (YiFeiXue, Nanjing, China, 1: 10 000) for 1 h at room temperature. Proteins were then visualized and detected using an electrochemiluminescence (ECL) system.
5
Protein Expression Analysis in Rat Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At day 7, rats in five groups were sacrificed by excessive anesthesia, and transcardially perfused with 4°C saline. Brain tissues were taken from cranial cavity and protein samples (n = 3) were extracted. Enhanced bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to determine protein concentration. The proteins that mixed with loading buffer were separated by 10% SDS-polyacrylamine gel and transferred to polyvinylidene fluoride membranes. Blots were probed with specific antibodies directed against GLUT1 (1:5000, Abcam), GLUT3 (1:8000, Abcam), MCT1 (1 μg/mL, Abcam), PDH (1:1000, Abcam), ACS (1:3000, Abcam), CS (1:2000, Abcam), and Beta actin (1 μg/mL, Abcam) at 4°C overnight. Subsequently, the membranes were gently washed and incubated with the fluorescent secondary antibodies at room temperature for 1 h. The signals of reactive bands were visualized by fluorescence scanner (LI-COR, Lincoln, NE, United States), and the expressions of targeted proteins in five groups were normalized against that of Beta actin.
6
Microglial Cell Protein Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction and subcellular fractionation were performed as we have previously described (44 (link)). For the preparation of whole-cell lysates, microglial cells were lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology) supplemented with cOmplete protease inhibitor cocktail tablets (5 mg/ml; Roche, Basel, Switzerland). Nuclear and cytoplasmic fractionations were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the instructions provided by the manufacturer. An enhanced BCA (bicinchoninic acid) protein assay kit (Beyotime Biotechnology) was used to measure the protein concentrations of the extracts.
7
Enzyme activity assays of insect pests
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extraction method of enzyme solution was according to the conventional method of Ding et al. (2021) (link). Three replicates were used for each strain, and each replicates contained thirty third instar nymphs (90 insects per strain).
The activities of carboxylesterase (CarE), glutathione-S-transferase (GST), and cytochrome P450 monooxygenase (P450) were determined according to conventional methods. CarE activity was evaluated according to the methods reported by Han et al. (1998) (link) and Ding et al. (2021) (link). Activity determination of GST and P450 refers to the practices of Kao et al. (1989) (link) and Aitio (1978) (link), respectively. According to Wen et al. (2021b) (link), the protein concentrations were measured using the Enhanced BCA (Bicinchoninic acid) Protein Assay Kit (Beyotime Biotechnology). The specific operation steps are inSupplementary Figure S2 .
The activities of carboxylesterase (CarE), glutathione-S-transferase (GST), and cytochrome P450 monooxygenase (P450) were determined according to conventional methods. CarE activity was evaluated according to the methods reported by Han et al. (1998) (link) and Ding et al. (2021) (link). Activity determination of GST and P450 refers to the practices of Kao et al. (1989) (link) and Aitio (1978) (link), respectively. According to Wen et al. (2021b) (link), the protein concentrations were measured using the Enhanced BCA (Bicinchoninic acid) Protein Assay Kit (Beyotime Biotechnology). The specific operation steps are in
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!