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29 protocols using bca protein assay kit

1

Protein Expression Analysis via Western Blot

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The cells were lysed in RIPA buffer (Aspen Biotechnology, Wuhan, China) supplemented with Protease Inhibitor Cocktail (Roche, NJ, USA) for 5 min. The concentration of total protein was determined using BCA protein assay kit (Aspen Biotechnology). Total protein samples (40 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were blotted onto polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking in 5% skim milk at room temperature for 1 hr, the PVDF membrane was incubated with primary antibodies at 4° C overnight. The PVDF membrane was washed three times with tris-buffered saline and tween 20 (TBST) then incubated with HRP-Goat anti Rabbit secondary antibodies (1:10000, Aspen) for 2 hr at room temperature. The blots were visualized with an enhanced chemiluminescence (ECL) kit (Aspen). β-actin was utilized a loading control. The primary antibodies for western blot were: HMGA1 (1:1000, Abcam, Cambridge, MA, USA), Bax (1:2000, Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (1:1000, Abcam), cleaved caspase-3 (1:1000, Abcam), β-actin (Abcam, 1:1000).
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2

Mouse Myocardial Protein Extraction

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This study was approved by the Animal Studies Subcommittee of our Institutional Review Board. We extracted proteins from 30 mg of C57BL/6J mouse myocardial tissue, adding 800 μL RIPA and 100× cocktail. Then, the tissue was ground twice (JingXin, shanghai, China; 10 Hz, 45 s) and lysed on ice for 10 min. The tissue suspension was centrifuged at 12,000 g for 20 min and the sediment was discarded. The protein concentration was determined by a BCA Protein Assay Kit (Aspen, Wuhan, China), according to the manufacturer’s instructions. The sample was added to 5× loading buffer and boiled at 100 °C for 10 min.
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3

Quantifying Liver NADPH Activity

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Liver homogenate was lyzed in mammal tissue protein extraction reagent. The extracted protein was then supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF) (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China). The samples were then centrifuged at 12,000 revolutions per minute for 15 minutes at 4°C. The supernatant was collected to quantify the protein concentration using a BCA protein assay kit (ASPEN Biotechnology Co. Ltd., Wuhan, China). Liver NADPH activity was measured using an NADPH activity quantification kit (Genmed Scientifics Inc., Shanghai, China).
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4

Quercetin Modulates CXCL1 and CXCL2 in Breast Cancer Cells

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MDA-MB-231 and MCF-7 cells were seeded in 6-well plates at 5.0 × 105 cells/well and treated with Quercetin (0, 20 and 80µM) for 24h. Then, cells in each well were photographed under 10x microscope. And the cell lysis was performed using RIPA lysis kit (AS1004, ASPEN, Wuhan, CHINA) and mixed with phenylmethanesulfonyl fluoride. Total protein quantification of the whole cell lysate was performed using BCA protein assay kit (AS1086, ASPEN, Wuhan, CHINA). The same total proteins were separated on 15% SDS-PAGE gel electrophoresis (AS1012, ASPEN, CHINA) and subsequently electrophoretically transferred onto a nitrocellulose membrane by electrophoresis (DTT-6C, DYCZ-400D, DYCZ-24DN, Beijing, CHINA). Membranes were blocked with 5% BSA for 2 h at room temperature, then incubated overnight at 4°C with primary antibodies, including CXCL1 (1:100 dilution, AB206411, ABCAM, UK) and CXCL2 (1:500 dilution, BS-1162R, BIOSS, Wuhan, CHINA). Membranes were washed 3 times, then incubated with HRP-Goat anti Rabbit (AS1107, ASPEN, Wuhan, CHINA). 1 h later, membranes were washed 3 times with tris buffered saline + Tween (TBST) for 10 minutes. Proteins were visualized by the addition of ECL (AS1059, ASPEN, Wuhan, CHINA), and membranes were scanned and imaged by the Fluor Chem FC3 system (Protein Simple, USA).
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5

Investigating Cellular Mechanisms with Diverse Reagents

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Pyruvate was supplied by Sigma (St. Louis, MO). Reverse transcript and RT-qPCR kits were obtained from ELK Biotechnology (Wuhan, China). Creatinine Assay Kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TRIpure Reagent was purchased from Aidlab Biotechnologies Co., Ltd. (Beijing, China). Total Protein Extraction Kit was obtained from Aidlab Biotechnologies Co., Ltd. (Beijing, China). BCA Protein Assay Kit was obtained from Aspen Biotechnology (Wuhan, China). Fetal bovine serum (FBS) and MEM/F12 (1 : 1) were supplied by Gibco (St. Louis, MO, USA). Penicillin-streptomycin combination was purchased from Genom (Hangzhou, China). Cell Counting Kit-8 (CCK-8) assay kit was obtained from Abcam (Shanghai, China). Apoptosis Assay Kit Annexin V-FITC-propidium iodide (PI) was purchased from BestBio (Shanghai, China). IOX2 was purchased from MCE (Shanghai, China). All oligonucleotide primers of the rat and human were synthesized by GeneCreate Biological Engineering Co., Ltd. (Wuhan, China). All synthetic concoctions were of analytical grade.
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6

Protein Expression Analysis in Canine Atrial Tissue

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We extracted proteins from 30 mg of canine atrial tissue or cultured cells. The protein concentration was determined by a BCA Protein Assay Kit (Aspen, as1086) according to the manufacturer's instructions. Proteins were separated on a 10% SDS-polyacrylamide gel and transferred to a 0.45 μM PVDF membrane by semidry transfer. The PVDF membranes were then blocked with 1% polyvinylpyrrolidone-40 and 0.05% Tween-20 for 30 minutes. The expression of target proteins was determined by incubating the membranes with the following primary antibodies overnight at 4°C: GAPDH (Servicebio, China, 1 : 1000), CD81 (Abmart, China, 1 : 1000), Rab27a (Servicebio, China, 1 : 1000), KCa3.1 (Proteintech, China, 1 : 1000), TSG101 (Servicebio, China, 1 : 1000), AKT (Servicebio, China, 1 : 1000), and p-AKT (Abmart, China, 1 : 1000). Visualization was performed on a chemiluminescence system after incubation with horseradish peroxidase-conjugated secondary antibodies (Proteintech, China, 1 : 3000) at room temperature.
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7

Western Blot Analysis of Autophagy Proteins

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Total protein was extracted from the cells using RIPA lysis buffer. Total proteins were quantified by the BCA protein assay kit (ASPEN). Next proteins (20 μg/lane) were dissolved by 10% SDS-PAGE and then electrotransferred onto a PVDF membrane (Millipore). After that, the membrane was incubated with specific primary antibodies, including anti-Beclin 1 (ab207612, 1:2,000, Abcam), anti-ATG5 (ab108327, 1:1,000, Abcam), anti-ERK (ab184699, 1:10,000, Abcam), anti-p-ERK (ab201015, 1:1,000, Abcam), anti-Bcl-2 (ab32124, 1:1,000, Abcam), anti-cleaved caspase 3 (ab32042, 1:500, Abcam), and anti-β-actin (ab6276, 1:5,000, Abcam), at 4°C overnight. Following incubation with the corresponding secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (ab7090, 1:5,000, Abcam), the protein blots were developed by ECL reagent. Band intensity was measured using ImageJ software (ImageJ, NIH).
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8

Hippocampal Protein Expression Analysis

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After all rats were euthanized with decapitation, the hippocampus was quickly dissected and stored at − 80 °C until use. Samples were homogenized in lysis buffer and then centrifuged, and the supernatants were collected. Finally, the protein concentration was determined by a BCA protein assay kit (Aspen Biological; China). Protein samples were separated on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Following blocking with blocking buffer for 1 h, the membranes were incubated at 4 °C overnight with rabbit anti-phospho-ERK1/2 (1:1000; CST; USA), rabbit anti-phospho-CREB (1:1000; CST; USA), mouse anti-Bcl-2 (1:1000; TDY Biotech; China) and rabbit anti-BAX (1:1000; Bioss; China) antibodies. Then, the membranes were incubated with HRP goat anti-mouse or goat anti-rabbit or rabbit anti-goat secondary antibody (Aspen Biological; China) for 1 h. Finally, the protein bands were scanned using AlphaEaseFC (AlphaInnotech, USA). In this experiment, we used traditional methods in Western blot, namely protein separation by electrophoresis, electric transfer and immune detection, where marker is prestained protein marker. Blots were cut prior to hybridisation with antibodies.
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9

Western Blot Analysis of Neuroinflammatory Markers

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Cardiac and dorsal root ganglion tissues were immersed in RIPA lysis buffer after being carefully washed with PBS. The protein concentration in the homogenates was measured with a BCA protein assay kit (Aspen, China, AS1086). The proteins were separated by 10% gel SDS–PAGE until the bromophenol blue ran off the gel and then transferred to a polyvinylidene difluoride membrane. Before incubation with primary antibodies, the membrane was blocked for 1 h at room temperature in 5% fat-free dry milk in PBS. The following primary antibodies were used: anti-rabbit caspase-1 (Abcam, USA, #ab179515), anti-rabbit NLRP3 (Alomono, Israel, #APR016), anti-rabbit ASC (Abcam, USA, #ab260043), anti-rabbit P2X3 (Invitrogen, USA, #PA5-115,707), anti-rabbit P2X7 (Abcam, USA, #ab93354), anti-rabbit P2X4 (Abcam,_USA, #ab168939), anti-rabbit P2X6 (ThermoFisher #PA5-87,659), and anti-rabbit IL-1β (ThermoFisher, USA, #M421B), anti-rabbit NGF (ThermoFisher #PA5-29,425), anti-rabbit GSDMD (Abcam, USA, #ab210070), anti-rabbit collagen I (Abcam, USA, #ab34710), anti-rabbit collagen III (Abcam, USA, #). The membrane was incubated with primary antibody overnight and then with HRP-conjugated goat anti-rabbit IgG (Sungene, China, #LK2001) for 30 min. The membrane was washed, and then the density of the protein bands was quantified using AlphaEaseFC software as described in previous studies [46 (link)].
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10

Western Blot Analysis of Protein Samples

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RIPA buffer containing the complete protease inhibitor was applied to lyse the tissues and cells. Lysates were centrifuged at 12,000 rpm for 5 min at 4°C. The concentrations of proteins in the supernatants were quantified using a BCA protein assay kit (Aspen, Canada, USA). Protein samples were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes. Then, the membranes were blocked with 5% nonfat dried milk for 1 h, followed by successive incubations with the primary antibodies (Table 3) and the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies. GAPDH served as a loading control. Protein bands were visualized using enhanced chemiluminescence (ECL) reagents and analyzed using an imaging system (QImaging, Surrey, Canada).
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