The total protein was extracted by total protein extraction kit (NanJing KeyGen Biotech Co., Ltd., China), the total protein concentration was measured by BCA method (NanJing KeyGen Biotech Co., Ltd., China), the 20 μg total protein was taken to electrophoretic separation by 10 % SDS-PAGE, transferred the protein to PVDF member, adding the fi rst antibodies (GAPDH 1:1000; TLR4 1:1000 and NF-κB 1:500) to culture overnight at 4 °C, washing by PBS, added HRP marked second antibody (1:1000) to culture for 1 h, adding enhanced chemiluminescence (ECL) to color, using Chemiluminescent imaging system (TANON 5200) to analysis the gray value by Imagel software.
Bca method
Manufactured by Keygen Biotech
Sourced in China
The BCA method is a colorimetric assay used for the quantitative determination of total protein concentration in a sample. It involves the reduction of Cu2+ to Cu+ by proteins in an alkaline medium, which then chelates with bicinchoninic acid (BCA) to produce a purple-colored complex that absorbs light at 562 nm. The intensity of the color is proportional to the protein concentration in the sample.
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Lab products found in correlation
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Sds sample buffer, by Keygen Biotech (2 mentions)
Anti β catenin, by Abcam (2 mentions)
Total protein extraction kit, by Keygen Biotech (1 mentions)
Immobilon p 0.45 μm, by Merck Group (1 mentions)
Ab15148, by Abcam (1 mentions)
Ab92574, by Abcam (1 mentions)
Ab133626, by Abcam (1 mentions)
Zb 2308, by ZSGB-BIO (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Anti e cadherin, by Proteintech (1 mentions)
Anti n cadherin, by Proteintech (1 mentions)
Anti gsk3β, by Abcam (1 mentions)
Anti c myc, by Abcam (1 mentions)
Anti vimentin, by Proteintech (1 mentions)
Anti p gsk3β ser9, by Abcam (1 mentions)
Anti akt2, by Abcam (1 mentions)
Anti akt1, by Abcam (1 mentions)
Anti prdx2, by Proteintech (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
8 protocols using bca method
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of Cell Proteins
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Cultured cells in an exponentially growing phase and frozen tissue samples were extracted with 1 ml of lysis buffer. The protein concentration of the lysate was determined by the BCA method (KeyGen, China). The extracts containing approximately 30 μg of protein were loaded onto 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrophoresed, and then transferred to polyvinylidence difluoride (PVDF) membranes (Immobilon-P. 0.45 μm, Millipore Corp., Bedford, MA, USA). The membranes were blocked with 5 % skimmed milk in PBST (phosphate-buffered saline with 0.1 % Tween 20) for 1 h and then incubated with primary antibody of rabbit anti-ECM1 antibody (Protein Group Inc, USA), rabbit anti-Vimentin antibody (ab92574, Abcam, USA), rabbit anti-E-cadherin antibody (ab15148, Abcam, USA), and rabbit anti-β-actin (ab133626, Abcam, USA) antibody at 4 °C overnight. After being washed three times with PBST buffer, the membranes were incubated with secondary peroxidase-conjugated antibody (ZB-2308, ZSGB-BIO) for 1 h at room temperature. After being washed three more times, the protein bands were detected by enhanced chemiluminescence (Pierce, Rockford, USA) according to the manufacturer’s instruction. β-actin antibody was served as a control to confirm equal loading. Densitometry index analysis of the bands was made using gel imagery system.
3
Protein Quantification and Western Blot Analysis
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Total proteins of cell samples were extracted with lysis buffer and then quantified using the BCA method (KeyGen Biotech, Jiangsu, China). The lysate was diluted in SDS sample buffer (KeyGen Biotech) for SDS-polyacrylamide gelelectrophoresis (PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Roche Applied Sciences,USA). The membranes were immunoblotted at 4 °C overnight with anti-PRDX2 (Proteintech, USA), anti-c-Myc(Abcam, UK), anti-p-c-Myc(S62) (Abcam), anti-p-c-Myc(T58)(Abcam), anti-E-cadherin (Proteintech), anti-Vimentin (Proteintech), anti-N-cadherin (Proteintech), anti-GSK3β (Abcam), anti-p-GSK3β (Ser9) (Abcam), anti-AKT(1/2) (Abcam), anti-AKT1 (Abcam), anti-AKT2 (Abcam), anti-p-AKT2 (Ser474) (Abcam) and anti-GAPDH (Proteintech) antibodies at appropriate dilution concentration, followed by incubation using the appropriate second antibodies for 2 h. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Image J software was used to analyze the grey value of the interest protein.
4
ELAVL1 and PI3Kδ Protein Analysis
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This work utilized RIPA buffer (Beyotime, Shanghai, China) for extracting total cellular proteins. BCA method (Keygen Biotech, Nanjing, China) was applied in determining protein content. After separating protein aliquots through SDS-PAGE, this study transferred on PVDF membranes (Millipore, MA, USA). Later, 5% defatted milk was later utilized to block membranes for a 1-h period. Blots were cultivated with anti ELAVL1 antibody (#12582, 1:1000, Cell Signaling Technology, Danvers, MA, USA) as well as PI3Kδ antibody (ab1678, 1:1000, Abcam, Cambridge, MA, USA) overnight under the temperature of 4˚C. Subsequently, this study adopted secondary antibody (Cell Signaling Technology) for additional 1-h incubation under ambient temperature. Signals were detected by enhanced chemiluminescence (Millipore). β-actin was used as internal reference.
5
Immunofluorescence and Immunoblotting Analysis
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Cells were cultured at a density of 1.5 × 105 cells/well on 8 mm coverslips in 12-well plates. After 48 hours, coverslips were fixed by ice-cold methanol, and incubated with primary E-cadherin (Abcam), Vimentin and β-catenin (Epitomics) antibodies prior to florescent-labeled secondary antibodies. Nuclear DNA was stained with 4′, 6-diamidino-2-phenylindole (DAPI) and coverslips were mounted with FluorSave reagent (CALBIOCHEM). Immunofluorescence images were taken by Olympus inverted fluorescence microscope and were outputted by PV10-ASW 1.7 viewer software. Immunoblotting was performed as follows: Proteins were extracted with lysis buffer and then quantified by the BCA method (KeyGen Biotech). Lysates were diluted in SDS sample buffer (KeyGen Biotech) prior to SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Roche Applied Sciences). Membranes were immunoblotted overnight at 4°C with anti-SOX17 (Millipore), anti-CyclinD1 (Abclonal), anti-C-myc, anti-DKK1 (Cell Signaling Technology), anti-E-cadherin (Abcam), anti-SOX2, anti-β-catenin, anti-Vimentin and anti-Slug and anti-N-cadherin antibodies (Epitomics), followed by the appropriate second antibodies. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Gel densitometry (Bio-Rad) was used to quantify immunoblot signals on exposed film.
6
Protein Quantification in BALF
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The BALF of each animal was collected to check the concentrations of protein utilizing the bicinchoninic acid (BCA) method (Keygen, China), with the instructions on the kit strictly followed.
7
Protein Extraction and Western Blot Analysis
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Briefly, the cells were collected and resuspended in RIPA buffer containing protease and phosphatase inhibitors. The mixture was vigorously shaken on ice for 30 min and centrifuged. The supernatant containing the proteins was collected and the total protein concentration in each sample was determined using the BCA method (KeyGen Biotech, Nanjing, China) at a wavelength of 562 nm. For the Western blot, 15 µg of protein per well were loaded into the gel. After the electrophoresis (80 V for 30 min, 120 V for 1 h) and transference (200 mA for 2 h), the membrane containing the proteins was blocked with 5% milk blocking solution for 1 h. The membrane was incubated overnight with anti-β-catenin (1:1000; Cat#ab32572), anti-MMP9 (1:1000, Cat#ab76003), and anti-cyclin D1 (1:1000; Cat#ab76003) from Abcam (Cambridge, UK), and anti-β-tubulin (1:1000; Cat#2128T) from Cell Signaling Technology (Danvers, MA, USA). The next day, the membranes were washed thrice with tris-buffered saline solution with Tween-20, and incubated with a horseradish enzyme labeled goat anti-rabbit secondary antibody (1:1000; Cat#ZB-2301) from ZSGB-BIO (Beijing, China). ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA) was used to evaluate the proteins and perform densitometry analysis of the target proteins using β-tubulin as control.
8
Protein Expression Analysis in BC Cells
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BC cells were lysed using RIPA buffer (KeyGEN, China) containing a protease inhibitor to extract total protein. The BCA method determined the protein concentration (KeyGEN, China). The films were developed using the ECL analysis method after conventional immunoblotting steps, such as electrophoresis, membrane transfer, blocking, and antibody incubation. This analysis aimed to assess the expression of proteins related to growth and metastasis (CDK4, CDK6, Cyclin D1, N-Cadherin, Vimentin, MMP9) in cells. GAPDH protein was used as an internal reference for optical density analysis.
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