Plant total protein extraction was conducted according to Zhou et al. [73 (link)] method. Two hundred mg of fresh sample were weighed into a pre-cooled mortar and ground with liquid nitrogen. According to Zhao et al. [74 ] method, a four-fold volume of phenol extraction buffer was added for ultrasonic cracking and an equal volume of tris balanced phenol (Solarbio, China) was added for centrifugation. The supernatant was removed into a centrifuge tube for later use, and the protein concentration was determined using the BCA protein kit (Biyuntian, China). Trypsin digestion protocol was done as follows: first, the extracted proteins were reduced with 5 mM dithiothreitol at 56 °C for 30 min. Next, each sample was alkylated with 11 mM iodoacetamide and incubated for 15 min at room temperature in the dark. Subsequently, the sample was diluted by adding 100 mM tetraethylammonium bromide (TEAB) to a urea concentration < 2 M. Finally, 2% trypsin was added for overnight digestion.
Bca protein kit
Manufactured by Beyotime
Sourced in China
The BCA protein kit is a colorimetric assay used for the quantitative determination of total protein concentration. It utilizes bicinchoninic acid (BCA) for the colorimetric detection and quantification of total protein in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (25 mentions)
Ripa lysis buffer, by Beyotime (20 mentions)
Ripa buffer, by Beyotime (6 mentions)
Gapdh, by Abcam (5 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (5 mentions)
Anti tlr4, by Cell Signaling Technology (4 mentions)
Anti hmgb1, by Cell Signaling Technology (4 mentions)
Ecl chemiluminescent method, by Merck Group (4 mentions)
Anti nf κb, by Cell Signaling Technology (4 mentions)
Anti p nf κb, by Cell Signaling Technology (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (4 mentions)
Immobilon p, by Merck Group (4 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Anti bcl 2, by Abcam (3 mentions)
Anti β actin, by Abcam (3 mentions)
Anti bax, by Abcam (3 mentions)
Quantity one software version 4, by Bio-Rad (3 mentions)
Ab8245, by Abcam (3 mentions)
141 protocols using bca protein kit
1
Plant Protein Extraction and Trypsin Digestion
2
Extraction and Characterization of Phellinus ribis
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Phellinus ribis was collected in Jinan, Shandong province (China). DEAE-cellulose 23 was acquired from Serva (Heidelberg, Germany) and Superdex 30 was purchased from Amersham Biosciences (Uppsala, Sweden). Hydrogen peroxide (H2O2, 30%, V/V), NaNO3, and ascorbic acid (Vc) were purchased from Guoyao (Shanghai, China). Standard monosaccharides, including d -mannose (Man), d -xylose (Xyl), d -glucose (Glc), d -galactose (Gal), larabinose (Ara), l -rhamnose (Rha), and d -glucuronic acid (GlcU), were purchased from sigma (St. Louis, MO, USA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and DMSO were purchased from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were acquired from Gibco (Carlsbad, CA, USA). Aβ25–35 was purchased from Abcam. Annexin V-FITC kit was from Bestbio (Shanghai, China) and JC-1 kit was from Kaiji (Nanjing, China). The BCA protein kit, TBST buffer, rabbit polyclonal antibodies against Cytc, rabbit polyclonal antibodies against caspase 3, and mouse monoclonal against caspase 3 were from Biyuntian (Wuhan, China); anti-GAPDH was from Solarbio (Beijing, China). All other reagents used in the experiment were of analytically pure agent grade.
3
Western Blot Analysis of Cartilage Proteins
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Western blot was performed following steps previously described [22 (link)]. Briefly, proteins were isolated from cartilage tissues of experimental rats or chondrocytes with RIPA lysis buffer and quantified using BCA protein kit (Both from Beyotime). 10 µg protein/lane was separated by 10% SDS-PAGE and transferred onto PVDF membranes. Subsequently, the membranes were incubated with primary antibodies against USP25, NLRP3, GSDMD-N, active caspase-1, TXNIP, and GAPDH at 4 °C overnight, followed by an incubation with horseradish peroxidase-conjugated secondary antibody for 1 h. Membranes were visualized with chemiluminescence reagent (Bio-Rad) with a ChemiDoc imaging machine (Bio-Rad).
4
Western Blot Analysis of NF-κB p65
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The concentrations of protein lysates were quantified using a BCA protein kit (Beyotime). Samples containing equal amounts of protein (20 μg) were mixed with loading buffer containing 5% 2-mercaptoethanol, heated for 10 min at 99 °C, and loaded onto a 10% SDS-PAGE gel. After electrophoresing, the proteins from the gel were then transferred onto polyvinylidene difluoride membranes. These membranes were blocked with 5% milk and 0.1% Tween 20 in Tris-buffered saline, and incubated overnight at 4 °C with anti-NF-κB p65 (Abcam), anti-LaminB1, anti-GAPDH and anti-β-Actin (Proteintech). Then, the appropriate horseradish peroxide-conjugated secondary antibody was added on the membranes at room temperature. Finally, the proteins were detected with chemiluminescent substrate.
5
Liver Tissue Protein Analysis
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The protein extracts of liver tissue were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and then transferred to polyvinylidene difluoride membranes (Millipore, United States). Protein concentration was quantified by the BCA method using the BCA protein kit (Beyotime Institute of Biotechnology, China). The proteins were probed with cyclooxygenase-2 (COX-2), caspase-3, Bax, Bcl-2, PARP and cleaved-PARP (Cell Signaling Technology, United States; 1:1,000), and detected by enhanced chemiluminescence. Secondary antibodies were used at a dilution of 1:10000. Protein bands were imaged using Bio-Rad Bio-Spectrum Gel Imaging System (Hercules, CA, United States), and the intensities were quantified by Photoshop CS6 software (Adobe, United States). All protein bands were normalized to that of GAPDH protein.
6
Western Blot Analysis of BEAS-2B Cells
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BEAS-2B cells were lysed with RIPA buffer containing protease inhibitor cocktail and phenylmethylsulfonyl fluoride. The supernatants were collected by centrifuged and total protein concentration was qualified using a BCA Protein Kit(Beyotime Biotechnology, Shanghai, China). Then equal quantities of protein samples were separated by 8% SDS-PAGE and transferred onto PVDF membranes(Meckmillipore,Germany) that were consequently blocked with 5% fat-free milk for 1hr. After that, the membranes were incubated with primary antibodies at 4 overnight. The primary antibodies were used as follows: anti-HMGB1(1:1000),anti-TLR4(1:1000),anti-NF-κB(1:1000), anti-p-NF-κB(1:1000), anti-Bcl-2(1:1000),anti-Bax(1:1000), and anti-βactin(1:1000)(Cell Signaling Technology, Danvers, MA,USA).Following extensively rinsing in TBST, the membranes were incubated with HRP-conjugated secondary antibodies and further detected using ECL chemiluminescent method (Millipore, Billerica, MA, USA). The blots were quantified with ImageJ.
7
Western Blot Analysis of EMT Markers
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The protein samples were obtained using RIPA lysis buffer. The protein concentration was detected using a BCA protein kit (Beyotime). A total of 50 µg of protein was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Inc.). The membrane was then blocked using 5% skim milk and incubated with E-cadherin (Rabbit monoclonal; dilution, 1:1,000; cat. no. ab1416; Abcam), N-cadherin (Rabbit polyclonal; dilution, 1:1,000; cat. no. ab18203; Abcam), Vimentin (Rabbit polyclonal; dilution, 1:1,000; cat. no. ab137321; Abcam), MMP-2 (Rabbit polyclonal, dilution, 1:300; cat. no. 10373-2-AP; Proteintech), MMP-9 (Rabbit polyclonal, dilution, 1:600; cat. no. 10375-2-AP; Proteintech), AKT (Rabbit polyclonal; dilution, 1:1,000; cat. no. ab8805; Abcam), p-AKT (phospho S473, Rabbit monoclonal; dilution, 1:1,000; cat. no. ab81283; Abcam), HMGB1 (Rabbit monoclonal; dilution, 1:1,000; cat. no. ab227168; Abcam) and GAPDH (Rabbit monoclonal; dilution, 1:1,000; cat. no. ab181602; Abcam) antibodies overnight at 4°C. After washing, the membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibodies (dilution, 1:5,000; cat no. ab190492; Abcam) for 2 h at 37°C. The protein bands were visualized using ECL (Pierce; Thermo Fisher Scientific, Inc.).
8
Western Blot Analysis of Lung Cell Proteins
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Lung tissues or cells were lysed with RIPA buffer containing protease inhibitor cocktail and phenylmethylsulfonyl uoride. The supernatants were collected by centrifuged and total protein concentration was quali ed using a BCA Protein Kit (Beyotime Biotechnology, Shanghai, China). Then equal quantities of protein samples were separated by 8% SDS-PAGE and transferred onto PVDF membranes(Meckmillipore,Germany) that were consequently blocked with 5% fat-free milk for 1hr. After that, the membranes were incubated with primary antibodies at 4℃ overnight. The primary antibodies were used as follows: anti-HMGB1 (1:1000; Cat#6893),anti-TLR4 (1:1000; Cat#14358), anti-NF-κB (1:1000; Cat#8242), anti-p-NF-κB (1:1000; Cat#3033), cleaved caspase-3 (1:1000; Cat#9661) (Cell Signaling Technology); anti-Bcl-2 (1:1000; Cat#ab182858),anti-Bax (1:1000; Cat#ab32503), and anti-βactin (1:1000; Cat#ab8227) (Abcam).Following extensively rinsing in TBST, the membranes were incubated with HRP-conjugated secondary antibodies and further detected using ECL chemiluminescent method (Millipore, Billerica, MA, USA). The blots were quanti ed with ImageJ.
9
METTL3 and HNRNPA2B1 Protein Analysis
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Proteins were isolated by RIPA buffer (Beyotime) and determined using a BCA protein kit (Beyotime). Thirty µg protein was loaded into a 10% sodium dodecyl sulfate polyacrylamide gel and then electrophoresed, transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% fat-free milk for 2 h at room temperature and incubated with primary antibodies against METTL3 and HNRNPA2B1 overnight at 4°C. Subsequently, the membranes were incubated with a horseradish peroxidase-conjugated immunoglobulin G secondary antibody for 2 h at room temperature. Enhanced chemiluminescence reagent was applied to the membranes for visualization. Protein bands were analyzed by Quantity One software version 4.62 (Bio-Rad). Original Western blot images were provided in Supplementary material 2.
10
Western Blot Analysis of IL-1β
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The DRG homogenates were dissolved in RIPA lysis buffer, protease inhibitor, and phosphatase inhibitor for protein extraction. The concentration of protein samples was estimated using the BCA protein kit (Beyotime, China). Protein samples (10 μg/lane) were separated using gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. PVDF membranes were blocked for 1 h at room temperature and incubated with primary antibodies overnight at 4℃ and their sources were as follows: IL-1β antibody The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, protein bands were visualized using the ECL kit (EMD Millipore). The band was quantified using Image J software.
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