Cell counting kit-8 (CCK-8) was purchased from Sigma-Aldrich. BCA protein quantitative kit was purchased from Boster (Wuhan, China). Mouse Nrf2, HO-1, SMAC, Cyt-C, Lamin-B, and β-actin monoclonal antibodies were purchased from Abcam. HRP-labeled goat anti-mouse IgG antibody and goat anti-rabbit IgG antibody were purchased from Santa Cruz. Commercial kit for the determination of lactate dehydrogenase (LDH) cytotoxicity was purchased from Thermo Fisher Scientific. Western blot electrophoresis and exposure system were purchased from Bio-Rad (CA, USA) and the automatic microplate reader was purchased from Yongchuang Medical Instruments (Shanghai, China). The other facilities used in this study included cell culture plate (Corning, USA), ultra-clean workbench (Yatai Kelong Instrument, Beijing, China), centrifuge (Shanghai Lu Xiangyi Centrifuge Instrument, China), cell counting plate (Germany), and carbon dioxide incubator (USA).
Bca protein quantitative kit
Manufactured by Boster Bio
Sourced in China
The BCA protein quantitative kit is a laboratory tool used to measure the total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) assay method to quantify the amount of protein present. The kit provides the necessary reagents and standard solutions to perform this analysis.
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Lab products found in correlation
Ab205719, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Cell culture plate, by Corning (1 mentions)
Cyt c, by Abcam (1 mentions)
Lamin b, by Abcam (1 mentions)
Goat anti rabbit igg antibody, by Santa Cruz Biotechnology (1 mentions)
Hrp labeled goat anti mouse igg antibody, by Santa Cruz Biotechnology (1 mentions)
Cell counting kit 8 cck 8, by Merck Group (1 mentions)
Ab97779, by Abcam (1 mentions)
Ab106629, by Abcam (1 mentions)
Enhanced chemiluminescence solution, by Merck Group (1 mentions)
Ab220807, by Abcam (1 mentions)
Protease inhibitor, by Boster Bio (1 mentions)
Ab8227, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Lipid peroxidation mda assay kit, by Beyotime (1 mentions)
Cellular glutathione peroxidase assay kit, by Beyotime (1 mentions)
Immulite 2000 xpi, by Siemens (1 mentions)
Ab128915, by Abcam (1 mentions)
Ab32370, by Abcam (1 mentions)
11 protocols using bca protein quantitative kit
1
Cell Viability and Protein Analysis
2
Western Blot Analysis of Protein Expression
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Cultured cells were collected by trypsinization, and then lysed with enhanced radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor (Boster, Wuhan, China), and then the protein concentration was determined by bicinchoninic acid (BCA) protein quantitative kit (Boster). The protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane and blocked with 5% bovine serum albumin at room temperature. Then diluted primary rabbit antibodies (SIRT1, ab220807; KLF4, ab106629; MMP2, ab97779; β-actin, ab8227; 1: 500; Abcam, Cambridge, MA, USA) were added into the membrane, followed by incubation at 4°C overnight. Next, horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (ab205719; 1: 2000; Abcam) was used to incubate the membrane for 1 hour at room temperature. The enhanced chemiluminescence solution (EMD Millipore, Bedford, MA, USA) was used to incubate the membrane at room temperature for 1 minute, after which it was then sealed with the plastic wrap, followed by X-ray film exposure for 5-10 minutes, development and fixation. Image J software was used to quantify the gray intensity of each band in Western blot analysis image, and β-actin was used as internal reference.
3
Lipid Peroxidation and Anti-Oxidant Assays
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The supernatant was collected after centrifugation (12000 rpm for 10 min) of homogenates prepared using a tissue grinder. Malondialdehyde (MDA) and glutathione peroxidase (GSH-Px), common biomarkers of lipid peroxidation and anti-peroxidation, respectively, were assayed in the supernatant using colorimetric and ultraviolet chromatometry methods with a Lipid Peroxidation MDA Assay Kit (#S0131, Beyotime, China) and Cellular Glutathione Peroxidase Assay Kit (#S0056, Beyotime, China), respectively, according to the manufacturers’ protocols.
Total protein was extracted using mammalian protein extraction reagent (Boster, China). Protein concentrations were determined with a BCA protein quantitative kit (Boster, China). Rat anti-mouse NF-κB p65 monoclonal antibody (#sc-71675, Santa Cruz Biotechnology, USA) diluted at 1:500 was used with horseradish peroxidase-conjugated rabbit anti-rat immunoglobulin (#AR1170, Boster, China) diluted at 1:2000. Protein bands were detected using an ECL chemiluminescence detection system (Amercontrol Biosciences, USA) and gel imaging system (Chemi Doc XRS+, USA).
Total protein was extracted using mammalian protein extraction reagent (Boster, China). Protein concentrations were determined with a BCA protein quantitative kit (Boster, China). Rat anti-mouse NF-κB p65 monoclonal antibody (#sc-71675, Santa Cruz Biotechnology, USA) diluted at 1:500 was used with horseradish peroxidase-conjugated rabbit anti-rat immunoglobulin (#AR1170, Boster, China) diluted at 1:2000. Protein bands were detected using an ECL chemiluminescence detection system (Amercontrol Biosciences, USA) and gel imaging system (Chemi Doc XRS+, USA).
4
Folate Quantification in Biological Samples
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Folate levels in serum, RBC and brain were measured with an automated chemiluminescence system (Siemens Immulite 2000 Xpi, Germany) using a competitive protein binding assay, according to the manufacturer’s instructions. This system detected all types of folate with a detection sensitivity limit of 0.8 ng/mL. Serum samples were obtained as described in section Animals and dietary treatment. Whole blood samples were collected and hematocrit was determined by automated hematology analyzer (URIT-2900VetPLUS, China), then cells were lysed in freshly prepared 0.5% (w/v) ascorbic acid solution (1:26, v/v) for 3 h at RT (15-28 °C) in the dark, and centrifuged to obtain the supernatant that later was used for folate assay. The folate concentration in RBC was calculated approximately by multiplying the concentration in whole blood by 100/ hematocrit.
Brain tissue was isolated and crushed with liquid nitrogen, then the homogenate (1:10, w/v, diluted with ice-cold normal saline) was centrifuged to obtain the supernatant that later used for folate assay. The folate concentration in brain was normalized to the protein concentration that was determined by a bicinchoninic acid (BCA) protein quantitative kit (Boster Biological Technology, China).
Brain tissue was isolated and crushed with liquid nitrogen, then the homogenate (1:10, w/v, diluted with ice-cold normal saline) was centrifuged to obtain the supernatant that later used for folate assay. The folate concentration in brain was normalized to the protein concentration that was determined by a bicinchoninic acid (BCA) protein quantitative kit (Boster Biological Technology, China).
5
Western Blot Analysis of Tumor Protein Expression
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The tumor tissue homogenate or cells were lysed with enhanced radio-immunoprecipitation assay (RIPA) lysate (Boster Biological Technology Co., Ltd., Wuhan, China) containing protease inhibitor for 20 min. The sample was centrifuged at 3000 g for 20 min and the supernatant was collected. The concentration of supernatant protein was detected by BCA protein quantitative kit (BOSTER). The protein was isolated by 10% SDS-PAGE. The isolated protein was transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% bovine serum albumin (BSA) for 2 h to block nonspecific binding, and respectively incubated using the primary antibodies p-ERK1/2 (Thr202/Tyr204) (#9101S, 1:1000, CST, Beverly, MA, USA), ERK1/2 (ab184699, 1:10,000, Abcam), and Bcl-xL (ab32370, 1:25; Abcam) at 4°C overnight. After washes by tris buffered saline-Tween20 (TBST), the sample was added with horseradish peroxidase (HRP) labeled secondary antibody and incubated for 1 h. Enhanced chemiluminescence fluid (EMD Millipore, Bedford, MA, USA) was used for development. Image-Pro Plus 6.0 (Media Cybernetics Inc., Silver Springs, MD, USA) was used to quantify gray scale of the band in each group in WB images, and GAPDH (ab128915, 1/10000, Abcam) was used as internal parameters.
6
Protein Extraction and Western Blot Analysis
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The Tissue or Cell Total Protein Extraction Kit (KeyGEN, Nanjing, China) was used to extract total proteins from cells and tissues, and the BCA Protein Quantitative Kit (Boster, China) was used to measure total protein levels. Samples were then stored at low temperature prior to use. Each sample was mixed with the loading buffer and heated to 100 °C for 10 min. Then, 25 μg of protein was added to each lane of the gel, followed by electrophoresis and membrane transfer. The membrane was sealed and incubated overnight at 4 °C with different primary antibodies (POSTN rabbit anti-human, Proteintech, China, 1:1000; PCNA rabbit anti-human, ABclonal, China, 1:1000; P-AKT rabbit anti-human, ABclonal, China, 1:1000; AKT rabbit anti-human, ABclonal, China, 1:1000; or PI3K rabbit anti-human, ABclonal, China, 1:1000). TBST was used to wash the membrane 3 times (5 min each) and goat anti-rabbit IgG (Boster, China, 1:8000) with horse-radish peroxidase was then added and incubated at room temperature for 1 h. TBST was used to wash the membrane 3 times (5 min each), and the ECL reagent was then added for visualization.
7
Assessing Brain Oxidative Stress
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Serum samples were obtained as described above in section Animals and dietary treatment, and brain tissue was prepared as described above in section Folate assay. Hcy and ROS levels in brain tissue were normalized to the protein concentration that was measured using a BCA protein quantitative kit (Boster Biological Technology, China). ROS levels were indicated by H2O2 concentration, HO• suppression ability and O2• – suppression ability.
8
Quantification of NF-κB and HIF-1α in Microglia
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After treating microglia as indicated, cells were lysed with RIPA buffer containing a protease inhibitor and phosphatase inhibitor (BOSTER, China) at a ratio of 100 : 1 : 1. After the cells were completely lysed, the supernatant was taken, and the protein concentration was measured using a BCA protein quantitative kit (BOSTER, China). The same amount of protein was separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Roche, Switzerland). Tween 20 (BOSTER, China) in Tris-buffered saline (BOSTER, China), containing 5% skim milk powder, was used to block the membrane for 2 h. Primary antibodies against NF-κB p65 (1 : 1400, ab16502, Abcam), Hif-1α (1 : 1000, 14179, Cell Signaling Technology), and GAPDH (1 : 1000, TA-08, ZSGB-Bio) were then incubated with PVDF membranes at 4°C overnight. Next, secondary antibodies (peroxidase-labeled) were incubated with the blots at 25°C for 1 h. The protein bands were observed using the enhanced chemiluminescence (ECL) system (GE Healthcare, UK), and relative protein expression was quantified using ImageJ software.
9
Transgenic Arabidopsis Protein Expression Analysis
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WT and WT35S::GmHXK2 transgenic Arabidopsis plants were grown on 1/2 MS medium containing 20 mg L-1 hygromycin. After 15 days of cultivation, total proteins of the seedlings were extracted from WT and transgenic Arabidopsis seedlings with a buffer consisting of 50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 0.2% (w/v) Triton X-100, 4% β-mercaptoethanol, 1 mM dithiothreitol (DTT), and 1% (v/v) protease inhibitor cocktail of which were then used for protein quantification using the BCA Protein Quantitative Kit (Boster). The protein samples (200 μg amounts) were electrophoresed in 8% SDS-PAGE and the gels were transferred to nitrocellulose membranes. The membranes were blocked with TBST buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) supplemented with 5% non-fat milk for 2 h and incubated with primary antibodies (Anti-GFP antibody, abcam, diluted at 1:1,000) in TBST buffer with 5% BSA overnight at 4°C. Afterwards, the membranes were washed three times (10 min each) with TBST buffer and incubated with the secondary antibodies (Goat Anti-Mouse IgG H&L (HRP), abcam, dilution at 1:1,000) for 2 h. After washing three times with TBST buffer, the membranes were incubated with a chromogenic agent using the Enhanced HRP-DAB Chromogenic Substrate Kit (Boster).
10
Western Blot Analysis of MMP-2, MMP-9, and CST1
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Enhanced RIPA cell lysis (Wuhan Boster Biological Technology., Ltd., Wuhan, China) containing protease inhibitors was used for cell lysis, and the protein concentration was determined using a BCA protein quantitative kit (Boster). The extracted protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes before being blocked with 5% BSA for 2 h to block non-specific binding. Diluted primary antibodies of MMP-2 (ab92536, 1:1000, Abcam, Cambridge, UK), MMP-9 (ab76003, 1:1000, Abcam), mouse anti-human CST1 (ab68329, 1:1000, Abcam), and GAPDH (ab9485, 1:2500, Abcam) were added for incubation at 4 °C overnight. Then the protein was washed and incubated with HRP labeled goat anti-rabbit secondary antibody (ab6721, 1:2000, Abcam) or rabbit anti-mouse secondary antibody (ab6728, 1:2000, Abcam) for 1 h, followed by treatment with ECL solution (EMD Millipore company, USA) at room temperature for 1 min. The membrane was exposed to X-Ray films for 5–10 min for color development. Image J analysis software (National Institutes of Health, NIH) was used for gray quantification of western blot images, with GAPDH as the internal reference. Each group had three parallel experiments.
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