The cells were harvested and washed once in PBS, then lysed on ice for 30 min with RIPA buffer (CST, 9806) and supplemented with 1% (v/v) protease inhibitor Cocktail (HY-K0010) and 1% (v/v) phosphatase inhibitors (Cocktail I, HY-K0021; Cocktail II, HY-K0022; and Cocktail III, HY-K0023) purchased from MCE (MedChemExpress, Shanghai, China). Western blotting was performed as described previously [13 (link)]. Protein concentrations were determined by BCA protein assay kit (Transgen Biotech), and equal amounts of proteins (15-50 μg/lane) were separated by SDS-PAGE (12% acrylamide running gel) and transferred to a nitrocellulose membrane (BioTrace™ NT, Pall Corp, FL, USA). The following antibody was used in this experiment: phospho-Bim (Thr56, Thr116) (PA5-64655, Thermo Fisher Scientific Shanghai, China); phospho-Bim (Ser69) (4581), p-JNK (9251S), p-ERK1/2 (9101S), cleaved caspase 3 (9664S), and Bim (C34C5) were all purchased from Cell Signaling Technology (Shanghai, China); ERK1/2 (abs130092), JNK1/2 (abs131832), and Actin (abs132001) were purchased from Absin (Shanghai, China). The Western blotting images were processed using Image J software (National Institutes of Health, Bethesda, MD, USA). The antibodies were diluted at the recommended ratio with Beyotime (P0256, Shanghai, China) diluent.
Bca protein assay kit
Manufactured by Transgene
Sourced in China
The BCA protein assay kit is a colorimetric detection and quantitation assay used to measure the total protein concentration in a sample. The kit utilizes the bicinchoninic acid (BCA) reaction to detect and quantify protein levels. The assay is based on the reduction of Cu2+ to Cu+ by protein in an alkaline medium, and the subsequent colorimetric detection of the Cu+ by bicinchoninic acid.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (3 mentions)
Biotrace nt, by Pall Corporation (3 mentions)
Gel imaging system, by Bio-Rad (2 mentions)
Lightshift chemiluminescent kit, by Thermo Fisher Scientific (2 mentions)
Ab81234, by Abcam (2 mentions)
Ab185645, by Abcam (2 mentions)
Ab134193, by Abcam (2 mentions)
Ab227073, by Abcam (2 mentions)
Ma3 1000, by Thermo Fisher Scientific (2 mentions)
Ab32572, by Abcam (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Bim c34c5, by Cell Signaling Technology (1 mentions)
Cocktail hy k0010, by MedChemExpress (1 mentions)
Claudin 1, by Santa Cruz Biotechnology (1 mentions)
Acsl4, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Occludin, by Zymo Research (1 mentions)
Claudin 2, by Zymo Research (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
30 protocols using bca protein assay kit
1
Western Blot Analysis of Phospho-Bim Signaling
2
Exosomal Protein Extraction and Detection
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The total proteins were extracted from exosomes or DF‐1 cells at 48 hours after transfection or exosome‐treated, and their concentrations were then determined using a BCA Protein Assay Kit (Transgen, China). Equal amounts of protein (5 μg of exosomal proteins and 50 μg of cell proteins) were transferred to nitrocellulose membranes after separation using 12% SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) (Beyotime, China) and blocked in 5% (w/v) skimmed milk at 25 ± 1°C for 1 hours, followed by incubation overnight at 4°C with the previous antibodies (anti‐CD9, CD63, 14‐3‐3ζ or GAPDH). After washing with TBST, the membranes were incubated with the secondary antibodies at room temperature for 1.5 hours. After another wash with TBST, the protein expressions on the membranes were detected using an ECLTM detection system (Bio‐Rad).
3
Protein Extraction and Western Blot Analysis
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KCs were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.5% Na-deoxycholate, and 0.1% SDS) containing the complete EDTA-free and PhosSTOP protease inhibitor cocktail (Bimake, Houston, TX, USA). The protein concentration was determined following the manufacturer’s protocol for the BCA Protein Assay kit (TransGen Biotech, Beijing, China). A total of 30–50 μg of protein was used for 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was transferred to a nitrocellulose membrane. The antibodies used for the western blot analysis are listed in Table S2 . Images were captured using the Tannon-5200 (Shanghai, China) and band density was analyzed using Image J software. GAPDH was used as a loading control for these specific proteins.
4
Protein analysis of rat ileums
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Total protein was extracted from rat ileums, and the concentration of total protein was determined by BCA Protein Assay kit (TransGen, Beijing, China). Western blotting was performed with the following primary antibodies: ACSL4 (Abcam), GPX4 (Abcam), FTH1 (Abcam), ZO-1 (Abcam), occludin (Zymed Laboratories), claudin-1 (Santa Cruz Biotechnology), and claudin-2 (Zymed Laboratories). β-Actin (Sigma) was used as loading control. For protein quantification, the density of Western blot bands was measured by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
5
Quantitative Analysis of Verticillium dahliae Secretome
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1 L of CDB liquid medium (NaNO3, 3.0 g/L, MgSO4·7H2O, 0.5 g/L, KCl, 0.5 g/L, FeSO4·7H2O, 0.01 g/L, K2HPO4, 1.0 g/L, sucrose 30 g/L) with 5% roots fragments (w/v) from 6-week-old cotton plants was inoculated with 1 ml of a V. dahliae spore suspension (105/ml) and subsequently cultured for 2 days in the dark at 25°C with shaking at 180 rpm. The culture filtrate was then centrifuged at 2,500 g for 30 min at 4°C, after which the supernatant was collected and passed through a 0.22 μm filter (Millipore, Suzhou, China). The proteins in the supernatant were precipitated with TCA as described in Wang et al. (2007 (link)). Protein concentrations were determined using a BCA™ Protein Assay Kit (TransGen Biotech, Beijing, China) with BSA as a standard. The mixture of proteins was analyzed via LC-MS/MS (Q Exactive, Bruker Daltonics K.K., Tokyo, Japan), combined with a BLAST search of the V. dahliae VdLs.17 genome database (http://www.broadinstitute.org/annotation/genome/verticillium_dahliae/MultiHome.html ).
6
Controlled Release Dynamics of Poloxamer 407 Hydrogels
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To evaluate the controlled release dynamics of the poloxamer 407 hydrogels used in the present study, a transwell assay (Cat: 3450, Costar, Corning Co., NYC, USA) was conducted using 17.0% (w/w) hydrogels. FGF-21, KGF-2, or KGF-2/FGF-21 mixtures (provided by the Key Laboratory of Biotechnology and Pharmaceutical Engineering, Wenzhou Medical University, Wenzhou, China) were separately dissolved in 17.0% (w/w) concentrations of cold poloxamer 407 solution, which were all made to a final concentration of 2 mg/mL. Then, 1 mL of poloxamer 407 protein solution was added into the upper compartment of transwell inserts with a 0.4 µm pore size, and the inserts were then incubated at 37°C for gelation. Once gelled, samples were placed in 6-well plates with 1 mL of 0.9% saline in the lower compartment. The saline solution in the lower compartment was analyzed for diffused FGF-21, KGF-2, or KGF-2/FGF-21 mixtures after 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 20 hours. Diffused protein was quantified via a BCA Protein Assay Kit (TransGen Biotech Co., Beijing, China).
7
Western Blot Analysis of Protein Expression
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Cells were harvested and lysed in RIPA lysis buffer (Thermo Fisher Scientific, USA) for extracting total proteins. The protein concentrations were measured by a BCA protein assay kit (Transgen Biotech, China), separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membranes. The membranes were then blocked by 10% nonfat milk and incubated with the corresponding antibody (CKIP-1, Akt, p-Akt, Gsk3β, β-catenin, p-β-catenin, Smurf1, and GAPDH; Abcam, Cambridge, MA, USA) diluted according to the instructions for 12 h at 4°C. After washing 3 times with PBS/0.1% Tween-20, membranes were incubated at room temperature for 2 h with horseradish peroxidase-conjugated secondary antibody (anti-mouse IgG and anti-rabbit IgG at 1:10000 dilution; Abcam). Protein expression levels were detected with ECL detection solution (Millipore, USA) and visualized with chemiluminescence detection system. The bands signal were analyzed using a gel imaging system (Bio-Rad).
8
CIITA Protein Expression in CRC
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CRC samples and their adjacent normal tissues of 7 patients were lysed according to the kit (PC101-PC104, Epizyme Biomedical Technology, Shanghai, China). Total protein concentration was quantified using a BCA protein assay kit (DQ111-01, TransGen Biotech Co., Ltd., Beijing, China). 50 μg of protein was separated by SDS–PAGE and was electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 3% BSA in Tris-buffered saline (TBS) containing 0.05% Tween-20 for 1 h. After blocking, the membranes were incubated with corresponding primary antibodies overnight at 4°C. The primary antibody for CIITA (#55099-1-AP; 1:1000) was purchased from Proteintech Group, Inc. (Wuhan, China). Membranes were washed by 1× TBST, followed by incubation with anti-Rabbit IgG-HRP (ZB-2301, Zhong Shan-Golden Bridge Biological Technology Co., Beijing, China) for 1 h. Immunoreactive bands were visualized by using Tanon 5500 (Tanon, Shanghai, China). Equal loading of proteins was verified by GAPDH (#60004-1-Ig; 1:3000, Proteintech Group, Inc., Wuhan, China).
9
Purification and EMSA of ERF114 Protein
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The coding region of the ERF114 gene was cloned into vector pET‐32. Recombinant plasmid with glutathione S‐transferases (His) tag was transformed into Escherichia coli BL21 (DE3) and then induced with 0.2 mM isopropyl‐β‐d ‐thiogalactoside (IPTG) at 20°C for 12 h. Cell pellets were collected and lysed by sonication in Tris‐HCl. His‐tagged protein was purified with His‐bind resin according to the manufacturer's instructions. Protein purification and quantification were performed using Ni‐NTA Resin (DP101; TransGen Biotech) and a BCA Protein Assay Kit (DQ111; TransGen Biotech), respectively. Next, 40‐bp probes within the indicated DNA fragment in the ERF114 promoter were labelled with biotin (Table S1 ). EMSA was conducted using a Light Shift Chemiluminescent Kit (Thermo Scientific).
10
Recombinant Expression and Purification of PevD1 and GhPR5 Proteins
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PevD1 (or its truncated fragments) without the predicted signal peptide (SP) and termination codon was inserted into the EcoRI and XbaI sites of the plasmid pPICZαA. The recombinant plasmid pPICZαA-pevd1 was linearized with the restriction enzyme PmeI and was subsequently transformed into Pichia pastoris KM71H for expression (Zhang et al., 2017b (link)). The GhPR5 gene without the predicted SP and the vacuole-targeting sequence was inserted into the BamHI and XhoI sites of the plasmid pRSETA (Invitrogen). The recombinant plasmid pRSETA-GhPR5 was transformed into E. coli BL21(DE3)pLysS for expression. The recombinant GhPR5 protein was expressed in inclusion bodies, and the purification and refolding of GhPR5 was carried out as previously described (González et al., 2017 ; Zhang et al., 2017c (link)). Proteins were detected by SDS-PAGE (RTD6110, Real-Times, Beijing), and were frozen at –80 °C in small aliquots until use. The protein concentration was measured using a BCA™ Protein Assay Kit (DQ111, TransGen Biotech). All primers used are shown in Supplementary Table S1 . The purified GhPR5 was used as an antigen for antibody production. Antibodies were produced in rabbits by the HuaAn Biotechnology Company.
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