A total of 100 mg hippocampal tissue from each sample was dissociated, homogenized followed by 10 min of centrifugation in a high-speed refrigerated centrifuge (15,521 × g; 4°C; Sigma-Aldrich; Merck KGaA). Subsequently, 20 µl supernatant was used to determine protein concentration using bicinchoninic acid assay protein assay kit (Beyotime Institute of Biotechnology), whereas the residual supernatant mixed with 5X SDS gel loading buffer. Western blot analysis was conducted as previously described (18 (link)). Proteins were separated by 12% SDS-PAGE (Beyotime Institute of Biotechnology), transferred onto polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA) before membranes were blocked and incubated by primary antibodies (VEGF, 1:500, cat no. AP0741; Bioworld Technology, Inc.; GAPDH, 1:1,000, cat no. 2118; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. Then membranes were developed with chemiluminescent assay kit (Invitrogen; Thermo Fisher Scientific, Inc. Waltham, MA, USA) and analyzed by ChemiDOC™ XRS+ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following incubation with horseradish peroxidase-conjugated secondary antibody (anti-rabbit-HRP IgG, 1:5,000; Cell Signaling Technology, Inc.) at room temperature for 1 h.
Bicinchoninic acid assay protein assay kit
Manufactured by Beyotime
Sourced in China
The Bicinchoninic acid assay protein assay kit is a colorimetric method for the quantification of total protein concentration in a sample. The kit utilizes the bicinchoninic acid (BCA) reaction to measure the reduction of copper ions (Cu2+ to Cu1+) by proteins in an alkaline environment. The resulting purple-colored reaction product is measured spectrophotometrically, allowing for the determination of the total protein content in the sample.
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Lab products found in correlation
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Sds page, by Beyotime (1 mentions)
Vascular endothelial growth factor (vegf), by Bioworld Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Las 4000, by Cytiva (1 mentions)
Enhanced chemiluminescence detection reagent, by Thermo Fisher Scientific (1 mentions)
Zo 1 33 9100, by Thermo Fisher Scientific (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
E cadherin, by Thermo Fisher Scientific (1 mentions)
N cadherin, by Thermo Fisher Scientific (1 mentions)
Ab8978, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
Ripa assay lysis buffer, by Beyotime (1 mentions)
Ab8245, by Abcam (1 mentions)
7 protocols using bicinchoninic acid assay protein assay kit
1
Hippocampal Protein Analysis via Western Blot
2
Western Blot Analysis of UDCA Effects
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M14 cells were plated in 6-well plates as described above following treating M14 cells with UDCA (0, 100, 200, and 300 µg/ml) at 37°C and 5% CO2 for 48 h, the cells were collected and washed twice. Proteins were extracted with RIPA buffer and centrifuged at 20,000 × g for 30 min at 4°C. Total protein concentration was determined using the bicinchoninic acid assay protein assay kit (Beyotime Institute of Biotechnology). Subsequently, the same amount of protein lysates (50 ug/lane) added with loading buffer were separated by 10% SDS-PAGE, which were then transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked with 5% fat-free milk at 4°C for 1 h. Soon afterwards, the membrane was incubated with the specific primary antibodies (diluted with ddH2O to 1:1,000) at 4°C overnight, and then proteins were visualized with horseradish peroxidase-coupled secondary antibodies (1:10,000) at room temperature for 1 h. Finally, the results were detected using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.).
3
Protein Expression Analysis in A549 Cells
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A549 cells were seeded at 1×106 cells/well in 6-well plates. Proteins were extracted from tumor tissues or A549 cells using a protein lysis buffer (RIPA; Beyotime Institute of Biotechnology). The concentration of protein was determined using a bicinchoninic acid assay protein assay kit (Beyotime Institute of Biotechnology). Proteins (25 μg/lane) were resolved using 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). The membranes were then incubated with primary antibodies (all at 1:1,000), followed by goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. no. ab181658; Abcam) at room temperature for 1 h. Proteins were visualized using Image Quant™ LAS 4000 (GE Healthcare Life Sciences) and quantified using Image J (version 1.46; National Institutes of Health). Anti-IL-21 (cat. no. ab5978), anti-IL-21R (cat. no. ab5980), anti-PD-L1 (cat. no. ab205921) and anti-Wnt (cat. no. ab28472) were purchased from Abcam. Anti-β-catenin (cat. no. 8480T), anti-cyclinD1 (cat. no. 3300T) and anti-GAPDH (cat. no. 5174S) antibodies were obtained from Cell Signaling Technology, Inc.
4
Quantitative Protein Analysis from Tumor
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Proteins from tumor tissues or cells were extracted according to the manufacturer’s protocol, and the protein concentrations were determined by using bicinchoninic acid assay protein assay kit (Beyotime, Beijing, China). An equal number of proteins were loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis for electrophoresis separation. The protein bands were transferred to polyvinylidene difluoride membranes and then incubated at 4°C overnight with primary antibodies. After that, the protein bands were incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The protein signal was detected using enhanced chemiluminescence detection reagents (Thermo Scientific, CA, USA), and glyceraldehyde 3-phosphate dehydrogenase was used as the internal reference.
5
Western Blot Analysis of Cell Signaling
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The cells were lysed with RIPA assay lysis buffer (Beyotime) on ice for 0.5 h. The whole cell lysates were then centrifuged at 4 °C for 20 min at 12,000 g. Protein concentrations were measured by bicinchoninic acid assay Protein Assay Kit (Beyotime). Denatured proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp, Billerica, MA, USA). The membranes were sealed with 5% skimmed milk or BSA for 60 min at room temperature and probed with primary antibodies to ZO-1 (33-9100, 1:1000, Thermo Fisher), E-cadherin (13-1700, 1:1200, Thermo Fisher), N-cadherin (33-3900, 1:1500, Thermo Fisher), Vimentin (ab8978, 1:1000, Abcam, Cambridge, UK), JMJD6 (sc-28,349, 1:800, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CCNB2 (sc-28303, 1:1300, Santa Cruz Biotechnology), p53 (sc-126, 1:2000, Santa Cruz Biotechnology), p-p53 (ab122898, 1:1000, Abcam), and GAPDH (ab8245, 1:1800, Abcam) overnight at 4 °C, and with the corresponding secondary antibody (ab205719, 1:3500, Abcam) for 60 min at room temperature. Finally, immunoblots were visualized by an ECL kit (32,209, Thermo Scientific). The intensity of the blotted bands was analyzed using ImageJ 1.8.0 (NIH, Bethesda, MA, USA).
6
Protein Expression Analysis Protocol
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Radioimmunoprecipitation assay lysis buffer (Beyotime) was used to extract proteins. Bicinchoninic Acid Assay Protein Assay Kit (Beyotime) was then implemented with reference to the instructions of guidebook to quantify protein. Next, the proteins were separated by SurePAGE gels (Beyotime) and transferred onto polyvinylidene fluoride membranes (Beyotime). The blocking solution (Beyotime) was added until the membrane was completely covered. After 2 h, the membrane was co‐incubated with primary antibodies for Bcl‐2‐like protein 4 (Bax) (ab32503, 1:1000, Abcam), B‐cell lymphoma 2 (Bcl‐2) (ab32124, 1:1000, Abcam), β‐actin (ab8226, 1:1000, Abcam), and SKA2 (ab75345, 1:1000, Abcam) overnight at 4°C, and then mixed with secondary antibody goat anti‐rabbit immunoglobulin G (IgG) (ab205718, 1:20000, Abcam) at 37°C for 2 h. Enhanced chemiluminescence (Beyotime) was then used for quantitative analysis of protein levels.
7
Western Blot Analysis of Brain Proteins
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The total proteins of brains were extracted by icecold cell lysis buffer (Beyotime, Shanghai, China). Bicinchoninic acid assay protein assay kit (Beyotime) was used to determine the total protein content. Equal amount of protein preparations were run on 8%-12% sodium dodecyl acrylamide-polyacrylamide gels, electro-transferred topolyvinylidine difluoride membranes, blotted overnight with primary antibodies and reacted with appropriated peroxidase-labelled secondary antibodies (Zhongshan Biotechnology Co., Beijing, China). Immunoreactive proteins were detected by the chemiluminescence assay (ECL Millipore, Billerica, Massachusetts, USA) using LAS-4000 chemiluminescence imaging system (Fujifilm, Tokyo, Japan). Quantitative analysis was performed using AlphaEasaFC analysis software (Alpha Innotech, San Leandro, California, USA) with horseradish peroxidase-conjugated monoclonal antibody against -actin serving as a control. 23 The primary antibodies included occludin (1:1000; Epitomics, Burlingame, California, USA), JAM-1 (1:1000; Cell Signalling, Danvers, Massachusetts, USA), AQP4 (1:1000; Cell Signalling), MMP-9 (1:1000; Santa Cruz Biotechnology, Santa Cruz, California, USA) and -actin (1:5000; Anbo Biotechnology Co., Ltd, San Francisco, California, USA).
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