Whole proteins were extracted from the Caco-2 monolayers using a previously described method. Protein concentrations were determined using BCA assay reagent (Beyotime). Samples (25 μg protein) were electrophoresed in an 8 or 10% polyacrylamide gel, and the proteins were then electrophoretically transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% BSA for 2 h at room temperature and was then incubated with the following primary antibodies overnight at 4℃: ZO-1 (1:500, Proteintech), Occludin (1:5000, Epitomics), Claudin-1 (1:1,000, Abcam), CYP1A1 (1:500, Proteintech), MLCK (1:1,000, Abcam), pMLC (1:1,000, Abcam), NF-κB p65 (1:500, Santa cruz), p-NF-κB p65 (1:1000, Cell signaling technology) and GAPDH (1:1,000, Goodhere Biotechnology). The membrane was subsequently incubated with a secondary antibody at room temperature for 1 h. Membrane imaging was performed using ECL western blotting detection reagent (Millipore) according to the manufacturer's instructions, and images were captured using an Image Station 4000R (Kodak).
Bca assay reagent
Manufactured by Beyotime
Sourced in China
The BCA assay reagent is a colorimetric detection kit used for the quantification of protein concentrations. It employs the bicinchoninic acid (BCA) method to detect and measure the total protein content in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Mammalian protease inhibitor cocktail, by Merck Group (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Occludin, by Abcam (1 mentions)
Cyp1a1, by Proteintech (1 mentions)
P mlc, by Abcam (1 mentions)
Ecl western blotting detection reagent, by Merck Group (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
Image station 4000r, by Kodak (1 mentions)
Claudin 1, by Abcam (1 mentions)
Bcl2 associated x (bax), by Bioss Antibodies (1 mentions)
Ripk1, by Bioss Antibodies (1 mentions)
Ripk3, by Bioss Antibodies (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
β actin, by Proteintech (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
Ab11939, by Abcam (1 mentions)
Sc 7210, by Santa Cruz Biotechnology (1 mentions)
Sc 13083, by Santa Cruz Biotechnology (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
16 protocols using bca assay reagent
1
Western Blot Analysis of Protein Expression
2
Placental Protein Extraction and Western Blot
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Total protein was extracted from placental tissues using radio-immunoprecipitation assay buffer (CST, United States) containing phenylmethylsulfonyl fluoride (CST, United States). Protein was quantified using the BCA assay reagent (CA# P0010, Beyotime, Nanjing, China) according to the manufacturer’s protocol. After denaturation, equal amounts of protein extracts were resolved by 10 or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene difluoride membranes (Roche, Mannheim, Germany). The membranes were blocked with 5% skim milk for 2 h at room temperature, and then incubated overnight at 4°C with primary antibodies against GRK2 (1:1,000, mouse; Santa Cruz, CA, United States), CC3 (1:1,000, mouse; Proteintech Corp., China), Bax (1:1,000, rabbit; BIOSS, China), Bcl2 (1:1,000, mouse; Santa Cruz, CA, United States), RIPK1 (1:1,000, rabbit; BIOSS, China), RIPK3 (1:1,000, rabbit; BIOSS, China), phosphorylated MLKL (p-MLKL) at serine 358 (1:1,000, mouse; BIOSS, China), and β-actin (1:5,000, mice; Proteintech, China). HRP-conjugated goat anti-rabbit (1:5,000, goat; Abbkine, United States) and anti-mouse (1:5,000, goat, Abbkine, United States) secondary antibodies were used to detect the respective protein bands. Signals were quantified using the Image Lab software (Bio-Rad, Hercules, CA, United States).
3
Ellagic Acid Regulates Caspase-3 Activity
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Following treatment with ellagic acid, liver tissue samples were acquired and homogenized via centrifugation at 12,000 × g for 10 min at 4°C. Total protein was measured using BCA assay reagent (Beyotime Institute of Biotechnology), and 5 µg protein was incubated with Ac-DEVD-pNA-caspase-3 activity kit (C1115; Beyotime Institute of Biotechnology) for 30 min at room temperature. The absorbance values were assessed at 405 nm using a spectrophotometer (GE Healthcare Life Sciences) according to the manufacturer's protocol.
4
Ellagic Acid Modulates Liver Protein Expression
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Following treatment with ellagic acid, liver tissue samples were acquired and homogenized for 10 sec at 12,000 × g for 10 min at 4°C. Total protein was measured using a BCA assay reagent (Beyotime Institute of Biotechnology), and then 50 µg protein was subjected to 10–12% SDS-PAGE and electrotransferred onto a polyvinylidene difluoride membrane (BD Biosciences, San Jose, CA, USA). The membrane was blocked in 5% non-fat milk in phosphate-buffered saline (PBS; pH 7.4) for 2 h, and subsequently incubated overnight at 4°C with the following primary antibodies: Anti-inducible nitric oxide synthase (iNOS; 1:400; sc-649; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-vascular endothelial growth factor (VEGF; 1:500; sc-13083; Santa Cruz Biotechnology, Inc.), anti-VEGF receptor 2 (VEGFR2; 1:3,000; ab11939; Abcam, Cambridge, UK) and β-actin (1:500; sc-7210; Santa Cruz Biotechnology, Inc.). Next, the membranes were washed with PBS and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:5,000; 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) at room temperature for 2 h. Protein expression in the samples was detected by Amersham ECL Prime western blotting detection reagent (GE Healthcare Life Sciences, Piscataway, NJ, USA) and analyzed using AlphaEase FC (FluorChem FC2) software (Cell Biosciences Inc., Santa Clara, CA, USA).
5
Protein Quantification in Cell Lysates
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Lysed cells and tissues were centrifuged at 13,000 x g for 30 min at 4°C after being incubated in cold RIPA buffer for 30 min. A BCA assay reagent (Beyotime) was used to calculate the protein levels of the lysates. Rabbit anti-occludin (1:1,000, Abcam), rabbit anti-ZO-1 (1:1,000, Abcam), and rabbit anti-β-actin (1:1,000, Abcam) were employed as the main antibodies. The chemiluminescent technique was used to identify proteins, and ImageJ was used to quantify them.
6
Nucleoprotein Extraction and Western Blot Analysis
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Nucleoproteins were extracted using a Nucleoprotein Extraction Kit (Sangon Biotech). Protein concentrations were determined according to the Bradford method using BCA assay reagent (Beyotime, Beijing, China). Samples (25 mg of protein) were loaded onto 8%–12% SDS-PAGE gels, and the proteins were then electrophoretically transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% BSA and then incubated overnight at 4°C with the following antibodies: anti-DJ-1 (1:400; Santa Cruz), anti-HIF-α (1:1,000; Abcam), anti-p-AKT (1:150; Cell Signaling), anti-PI3K-p110α (1:500; Cell Signaling), and anti-PCNA (1:500; Cell Signaling). After the membranes were washed, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling) at room temperature for 1 hour. The proteins were visualized using an enhanced chemiluminescence (ECL) Kit (Amersham Life Sciences, Arlington Heights, IL, USA) and exposed using a Chemiluminescence Imaging System (Fusion Solo S, Vilber, France).
7
Western Blot Analysis of Tight Junction Proteins
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The cells were washed twice with ice-cold PBS, and lysed in cold RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1ug/ml APMSF, 1.0 mM sodium orthovandate) containing mammalian protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined according to the Bradford method using BCA assay reagent (Beyotime). Samples (25 μg protein) were loaded onto SDS-PAGE gels and the gels were transferred to PVDF membranes (Millipore) after electrophoresis. Membranes were blocked by 5% bovine serum albumin (BSA) in TBST (50 mM Tris-HCl PH 7.5, 140 mM NaCl, 0.1% Tween) and then incubated with the following primary antibodies at 4℃ overnight: mouse monoclonal anti-ZO-1 (1:1000), rabbit monoclonal anti-Occludin (1:1000), rabbit polyclonal anti-Claudin-1 (1:1000), rabbit polyclonal anti-OS-9 (1:1000), rabbit polyclonal anti-GAPDH (1:1000). The membranes were then washed three times and incubated with HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibody. Membrane imaging was performed using an ECL kit (Thermo) according to the manufacturer's instructions.
8
Protein Extraction and Quantification
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Whole proteins were extracted from the U266 and KMS27 monolayers using RIPA buffer (strong) according to the manufacturer’s instructions. The protein concentrations were quantified using BCA assay reagent (Beyotime). MCM2 (D7G11) XP® Rabbit mAb (1:200, Cell Signaling Technology, #3619), MCM3 (D47B6) Rabbit mAb (#4003), DHFR (E6L1H) rabbit monoclonal antibody (mAb) (1:200, Cell Signaling Technology, #43497), and GAPDH monoclonal antibody (1:100, Proteintech, Cat No. 60004-1-Ig).
9
Protein Extraction and Analysis from Liver Tissues
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Liver tissues (n = 4) were homogenized in liquid nitrogen and the total proteins were extracted with RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing 1 mM PMSF (KeyGEN BioTECH, Nanjing, China) and 1% phosphatase inhibitor. The samples were centrifuged at 12,000 g, 4 °C for 10 min., and the supernatants liquid was collected as protein samples. The protein content was determined by using BCA assay reagent (Beyotime Biotechnology, Shanghai, China) in accordance with the manufacturer’s instructions. All of the protein samples were adjusted to an equal concentration with 5× loading buffer (Beyotime Biotechnology, Shanghai, China) for protein denaturation [19 (link)]. Briefly, the total protein was isolated by 10% SDS-PAGE gel (Beyotime Biotechnology, Shanghai, China) and then transferred onto Immobilon®-P transfer membranes (Millipore Corp, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China), and then primary antibody against phospho-mTOR (Ser2448), mTOR, phospho-AKT (Ser473), AKT, and β-actin (Cell Signaling Technology Inc., Danvers, MA, USA) was applied overnight at 4 °C. After incubating with the secondary antibody at room temperature for 2 h, the membrane was detected by chemiluminescence (Applygen Technologies Inc., Beijing, China) [21 (link)].
10
Western Blot Analysis of Notch Signaling
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Tissues and cells were homogenized in cold RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 μg/mL PMSF, 1.0 mM sodium orthovanadate, and 1× mammalian protease inhibitor cocktail; Sigma-Aldrich, Shanghai, China). Protein was quantified by the Bradford method using the BCA assay reagent (Beyotime, Shanghai, China). Equal amounts of protein were loaded into SDS-polyacrylamide gels and transferred onto PVDF-Plus membranes. After blocking in 5% fat-free milk for 1 h at room temperature, the membranes were incubated overnight at 4 °C with the following primary antibodies: NICD2 (ab-52302, Abcam, UK), Hes5 (sc-25395, Santa Cruz, Dallas, CA, USA), and GAPDH (sc-32233, Santa-Cruz, Dallas, CA, USA). The primary antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG, or goat anti mouse IgG secondary antibody for 1 h at room temperature and detected by the use of a chemiluminescence system (Beyotime, Shanghai, China) and imaging system (Kodak Gel Logic 4000R Imaging System, Carestream, Rochester, NY, USA). A semiquantitative densitometry analysis of the bands were performed using the Kodak Gel Logic 4000R Imaging System (Carestream, Rochester, NY, USA). Protein expression was normalized to the same sample’s expression of GAPDH.
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