Proteins were expressed by adding 0.4 mM isopropyl-β-d -thiogalactoside (IPTG) to cultures of E. coli Rosetta(DE3) cells containing pDEST17 constructs grown in LB medium for 6 h at 37°C, 200 rpm. After overnight culture, bacterial cell suspensions were sonicated and centrifuged (12,000 rpm, 10 min), and pellets were washed twice in a washing buffer (50 mM Tris-HCl, 0.05 M EDTA [pH 8.0] containing 2% deoxycholate [DOC], 1 M urea) before resuspension in 50 mM Tris-HCl, 1 mM EDTA (pH 8.0) containing 15 mM DTT, 8 M urea. To reconstitute proteins from inclusion bodies, dissolved proteins were dialyzed overnight against 200 mM Tris-HCl (pH 10.0) containing 8 M urea, 20 mM l -arginine, using a pump to add water into the buffer slowly until it was diluted 10 times, and then against 20 mM Tris-HCl (pH 8.0) containing 20 mM NaCl. Endotoxins were removed during the purification process by the addition of 2% DOC. Protein concentration was determined using modified bicinchoninic acid (BCA) protein assay kits (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions.
Bca protein assay kit
Manufactured by Sangon
Sourced in China
The BCA Protein Assay Kit is a colorimetric-based method for the quantitative determination of protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) reaction, which produces a purple-colored complex that absorbs light at a wavelength of 562 nm. The intensity of the color is proportional to the protein concentration in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (20 mentions)
Tissue or cell total protein extraction kit, by Sangon (9 mentions)
Ripa buffer, by Beyotime (8 mentions)
Ripa lysis buffer, by Sangon (7 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
β actin, by Cell Signaling Technology (6 mentions)
Beyoecl moon, by Beyotime (6 mentions)
Total protein extraction kit, by Sangon (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Ripa buffer, by Sangon (4 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (4 mentions)
Ecl kit, by Merck Group (4 mentions)
Protease inhibitor, by Beyotime (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Microplate reader, by Thermo Fisher Scientific (4 mentions)
Enhanced chemiluminescence reagent, by Sangon (3 mentions)
Protease inhibitor cocktail, by Sangon (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Gapdh, by Proteintech (3 mentions)
Ripa lysis buffer, by Solarbio (3 mentions)
235 protocols using bca protein assay kit
1
Protein Expression and Purification from E. coli Inclusion Bodies
2
Protein Expression Analysis in Iron Metabolism
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Proteins were extracted, and the concentration was determined according to standard protocols of protein extraction and BCA protein assay kits, respectively (Sangon Biotech, Shanghai, China). The total protein in the supernatants (30 μg/well) was separated by 10% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with anti-hepcidin (1 : 5000; no. ab190775, Abcam, Cambridge, MA, USA), anti-TfR1 (1 : 5000; no. ab269514, Abcam), anti-Ferritin (1 : 5000; no. ab75973, Abcam), anti-DMT1 (1 : 1000; no. ab157208, Abcam), anti-ALK2 (1 : 500; no. ab262699, Abcam), anti-BMP6 (1 : 1000; no. ab155963, Abcam), anti-SMAD1/5/8 (1 : 1000; orb162781, Biorbyt, UK), and anti-GAPDH (1 : 5000; no. ab8245, Abcam) antibodies. After three washes with TBST, the immunoblots were incubated with alkaline phosphatase-labeled goat anti-rabbit antibodies (1 : 1000, Cell Signaling Technology, USA). The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Sangon Biotech). The blots were semiquantified by ImageJ software 1.47 (National Institutes of Health, Bethesda, MD, USA).
3
Protein Expression Analysis of Stem Cells
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The cells (hWJMSCs, hBMSCs, and chondrocytes) and tissues were extracted used the Total Protein Extraction Kit (Sangon Biotech). Next, BCA Protein Assay kits (Sangon Biotech) were used to determine the protein concentration. The total protein was separated with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to P membrane, and blocked in 5% skimmed milk. Subsequently, the membrane was incubated overnight with exosome-associated primary antibodies (CD63, CD9, and TSP70) as well as the following primary antibodies: ITGB1, TGF-β, p-Smad2/3, Smad2/3, Smad6, COLL-II, and β-actin. The next day, all membranes were washed with PBST three times, and then incubated with the appropriate secondary antibody for 1 h. An enhanced chemiluminescence reagent (Sangon Biotech) was used to visualize the immunoreactive bands. The strength of each band was detected using ImageJ software (National Institutes of Health, USA).
4
Western Blot Analysis of Notch Pathway Proteins
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Proteins were extracted from rat spinal cords and PC-12 cells with RIPA buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration was determined according to the standard protocols of BCA protein assay kits (Sangon Biotech, Shanghai, China). Protein samples (40 µg) were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked in 5% skim milk for 1 hour at room temperature, the membranes were incubated with primary antibodies against EZH2 (1:1,000; Abcam, Cambridge, MA, USA), Notch1 (1:1,000; Abcam, Cambridge, MA, USA), Notch3 (1:1,000; Abcam, Cambridge, MA, USA), Notch4 (1:1,000; Abcam, Cambridge, MA, USA), PS-1 (1:2,000; Abcam, Cambridge, MA, USA), Hes1 (1:1,000; Abcam, Cambridge, MA, USA), or β-Actin (1:2,000; Abcam, Cambridge, MA, USA). After incubation at 4 °C overnight, The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 hours at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Beyotime, Beijing, China). Each band was semi-quantified using ImageJ (version 1.47; National Institutes of Health, Bethesda, MD, USA).
5
Laccase Activity Determination Protocol
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The laccase activity was measured using the method described in the study by Fang et al. (2015b) with slight modi cations. The mycelia were cultivated in liquid culture and shaken for 4 days. The supernatants were collected to detect the laccase activity. Reactions with 10 μl 200 μM ABTS, 40 μl supernatants and 150 μl 50 mM NaAc buffer (pH 5.0) were conducted. After reaction for 5 min at 25 ℃, the mixtures were transferred to a water bath and boiled for 5 min. The absorbance of the reaction was measured at 420 nm. Reactions using heat-treated supernatants were used as the control. The concentration of proteins in the supernatants was detected by BCA protein assay kits (Sangong Biotech, Shanghai) according to the manufacturer's guidelines. One unit of laccase activity was calculated as the enzyme amount required for an OD increase of 1 after 1 min of reaction of each supernatant. The laccase activity was de ned as units of laccase activity per mg protein.
6
Purification of PcoI and LqqP Proteins
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The LqqP and PcoI proteins were expressed as His6-fusions and purified by affinity chromatography. The coding region of pcoI was cloned into plasmid pET30a (Table S1 ) using primers listed in Table S2 . The coding region of lqqP was cloned into plasmid pClod TF (Table S1 ). The genes were then expressed in E. coli BL21(DE3) (Table S1 ). The His6-fusion proteins were purified from 400 mL E. coli BL21(DE3) carrying the pET30a or pClod TF plasmid derivatives using Ni-NTA resin (GE Healthcare, Shanghai, China). The strains were grown to OD600 0.4 at 37 °C, and then 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma, USA) was used to induce gene expression at 28 °C for 4 h to obtain PcoI or at 15 °C for 24 h to obtain LqqP. The concentration of purified proteins was determined by BCA protein assay kit (Sangon Biotech, Shanghai, China). Protein purity was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).
7
Western Blot Analysis of DNMT Proteins
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CAR-T cells were treated with various concentrations of DAC for different times. Total proteins were extracted and protein concentration was detected by BCA Protein Assay Kit (Sangon Biotech, Hangzhou, China) according to the manufacturer's manual. An equal amount of total proteins was subjected to Western blotting as previously described (28 (link)). The membranes were visualized using the ECL kit (Biological Industries, Beit Ahemeq, Israel) and images were obtained by the ChemiDoc MP Imaging System (Bio-Rad, Hercules, USA). The primary antibodies used in this study included anti-DNMT1, anti-DNMT3A antibodies, and anti-β-actin was used to ensure equivalent loading of whole-cell protein, which were purchased from Cell Signaling Technology (Beverly, MA, USA).
8
RAW 264.7 Cells Response to 5-HMF and LPS
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RAW 264.7 cells (1 × 106 cells/each well in 6-well plates) were pre-treated with 0, 31.5, 63.0 and 126.0 μg/mL of 5-HMF for 6 h and then stimulated by 1 μg/mL of LPS for 18 h. The cells were washed with cold PBS and then total protein extraction was conducted with the RIPA lysis buffer (Beyotime, China) on ice. The protein concentration was assessed with a BCA protein assay kit (Sangon Biotech, Shanghai, China). Western blot analysis was carried out using the standard protocol: the quantified proteins (15 μg) were subjected to electrophoresis on 12% SDS-PAGE gels for 90 min and then transferred to PVDF membranes for 2 h (Mini Gel Tank, Invitrogen, Gaisburg, MD, USA). The PVDF membranes were blocked in 5 % skimmed-milk in TBST for 2 h and washed with TBST three times and then they were incubated with primary rabbit monoclonal antibodies against p38, JNK, ERK1/2, p-p38, p-JNK, p-ERK1/2, Akt, p-Akt, mTOR, p-mTOR, IκBα, p65, p-IκBα, p-p65 and β-Actin at 4 °C overnight. Next, they were washed three times with TBST and incubated with an anti-rabbit HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA) at a 1:8000 dilution at room temperature for 1 h. The proteins were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA) with the ChemiDoc-It Imaging System (UVP, Upland, CA, USA).
9
Quantitative Western Blot Analysis
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Transfected cells or homogenized tumor xenografts were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology), and then subjected to centrifugation 10,000 × g at 4°C for 5 min to harvest the supernatant and extract total protein. Total protein quantification was performed with a BCA Protein Assay kit (Sangon Biotech Co., Ltd.). Equal amounts of protein (30 μg) were subjected to 10% SDS-PAGE. Then, the targeted proteins were transfected to PVDF membranes. After blocking in 5% non-fat dried milk diluted in TBS-0.05% Tween-20 at room temperature for 2 h, the membranes were incubated overnight at 4°C with primary antibodies targeting HDGF (cat. no. ab128921; 1:1,000; Abcam) or GAPDH (cat. no. ab8245; 1:1,000; Abcam). The goat anti-mouse (cat. no. ab205719; 1:5,000; Abcam) and goat anti-rabbit (cat. no. ab6721; 1:5,000; Abcam) IgG horseradish peroxidase-conjugated secondary antibody was incubated with membranes at room temperature for 2 h. Subsequently, positive bands were detected using an ECL Advance western blotting detection kit (Cytiva). Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.) was used for densitometry.
10
Protein Extraction and Western Blot Analysis
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Total protein was isolated from muscle satellite cells using a Tissue Total Protein Extraction Kit (Sangon, Shanghai, China) and concentrations were determined using a BCA Protein Assay Kit (Sangon, Shanghai, China). Then, proteins were separated in 10% polyacrylamide gel, moved to polyvinylidene difluoride (PVDF), blocked with 2% BSA for 60 min at room temperature, and then, incubated with primary antibodies overnight at 4 °C. The ACC, pACC (Ser79), GSK3β and pGSK3β (Ser9) antibodies were from Cell Signaling Technology (Danvers, MA, USA). AMPK alpha, pAMPK alpha (Thr172) and LC3B-II antibodies were from Affinity Biosciences (Cincinnati, OH, USA). HRP-goat anti Rabbit, GAPDH and p62 antibodies were from Abcam (Cambridge, UK). Immunoblots were performed over two hours at room temperature using HRP-labeled goat anti-rabbit IgG, followed by detection using an ECL detection system (Beyotime, Shanghai, China).
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