Cells were collected and proteins were extracted with radioimmunoprecipitation assay lysis buffer (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The protein concentration was determined by the BCA protein assay (Shanghai Yeasen Biotech Co., Ltd., Shanghai, China). Protein samples (40 µg per lane) were separated by 10% SDS-PAGE and then electro-transferred onto nitrocellulose membranes for 90 min. The membranes were blocked for 1 h with 5% skimmed milk at room temperature and incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies. Primary antibodies used were as follows: Specific to FOXP3 (cat no. 236A/E7; 1:500; anti-mouse; Abcam, Cambridge, MA, USA), CTNNB1 (cat no. 8480; 1:1,000; anti-rabbit), E-cadherin (cat no. 3195; 1:1,000; anti-rabbit), N-cadherin (cat no. 13116; 1:1,000; anti-rabbit), Vimentin (cat no. 5741; 1:1,000; anti-rabbit), GSK3β (cat no. 12456; 1:1,000; anti-rabbit) and GAPDH (cat no. 2118; 1:5,000; anti-mouse) (all purchased from Cell Signaling Technology, Inc., Danvers, MA, USA). Secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit/mouse immunoglobulin G (cat no. 111-035-003/115-035-003; 1:5,000; Jackson Laboratory, Bar Harbor, ME, USA). The reactions were visualized using enhanced chemiluminescence kit (Merck KGaA, Darmstadt, Germany) and a gel imaging system.
Bca protein assay
Manufactured by Yeasen
Sourced in China
The BCA protein assay is a colorimetric detection and quantification method for determining the total protein concentration in a sample. It is based on the reduction of copper ions by proteins in an alkaline medium, which results in the formation of a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Ctnnb1, by Abcam (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Keygen Biotech (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Hitrap sp ff, by GE Healthcare (1 mentions)
Lysis buffer for wb ip assays, by Yeasen (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
4 protocols using bca protein assay
1
Western Blot Analysis of EMT Markers
2
Protein Expression Analysis of NLRP3 Inflammasome in HSCs
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HSCs were lysed in radioimmunoprecipitation (RIPA) lysis buffer (Beyotime Institute of Biotechnology) and the concentrations of the proteins were measured using a BCA protein assay (Shanghai Yeasen Biotechnology Co., Ltd.). Proteins (40 µg/lane) were separated via 10% SDS-PAGE and were transferred to PVDF membranes, which were then blocked with 5% skimmed milk for 1 h at room temperature. Subsequently, the membranes were incubated at 4˚C overnight with primary antibodies (all 1:1,000) against NLRP3 (cat. no. 15101S), IL-1β (cat. no. 12703S), caspase-1 (cat. no. 3866T), α-SMA (cat. no. 19245T), COL1A1 (cat. no. 72026T), TIMP1 (cat. no. 8946S) and GAPDH (cat. no. 5174T; all from Cell Signaling Technology, Inc.). The membranes were washed three times with TBS-0.2% Tween 20 before incubation with goat anti-rabbit HRP-conjugated secondary antibody (1:3,000; cat. no. 7074S; Cell Signaling Technology, Inc.) for 1 h at room temperature. The expression levels of the targeted proteins were determined using a Bio-Rad ChemiDoc MP imaging system (Bio-Rad Laboratories, Inc.). The bands were detected using an enhanced chemiluminescence reagent (EMD Millipore). The grayscale values of the membranes were semi-quantified using ImageJ software (version 1.52r; National Institutes of Health). GAPDH was used as an internal control.
3
Tau Protein Purification and Characterization
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The above bacteria were harvested and resuspended in SP buffer (20 mM Na2HPO4·12H2O, 20 mM NaH2PO4·2H2O, 1 mM EDTA, 0.5% β-Mercaptoethanol, 1 mM PMSF, pH 6.8) and were then broken by sonication on ice. The supernatants were filtered, loaded onto a cation exchanger HiTrap SP FF (GE Healthcare), and washed with SP buffer. A linear gradient of salt (0–400 mM NaCl) in the same buffer as used to elute the Tau protein. BCA Protein Assay (Yeasen) and Coomassie brilliant blue staining of 10% SDS-PAGE were used to estimate protein concentration and purity, respectively. The purified proteins were dialyzed three times with 10 mM HEPES buffer (10 mM HEPES, 100 mM NaCI, 0.5% β-ME, pH7.4), concentrated with a 10K Amicon centrifugal filter (Millipore) for kinetic analysis, and transferred to −80 °C for long-term storage.
4
Western Blot Analysis of EMT Markers
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Total protein was extracted using Lysis Buffer for WB/IP Assays (Yeasen Biotechnology). Protein concentrations were determined using the BCA protein assay (Yeasen, China) according to the manufacturer's instructions. An equivalent amount of protein (20 μg) was subjected to 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a PVDF membrane (0.45 μm, Merck Millipore, Germany). The membranes were blocked for 30 min with 5 % skimmed milk powder in TBST at room temperature. The blocked membranes were individually incubated overnight at 4 °C with primary antibodies against Fibronectin (1:1000,Abcam, Ab268021), E-cadherin (1:1000, CST, 3195T), Vimentin (1:1000, CST, 5741T),β-actin(1:1000,servicebio,GB5003-100). Then, the membranes were washed and incubated with secondary antibodies (1:5000, CST, USA) for 1 h at 37 °C.The chemiluminescence signal was detected using Enlight buffer (Engreen, China). The optical densities of the bands were quantified using Image Lab software (Bio-Rad, USA) and normalized to GAPDH as internal control.
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