The cells were lysed for 30 min using RIPA reagent (Beyotime, China) containing protease inhibitors (Beyotime) on ice, and then the supernatant was collected by centrifugation (12,000 rpm, 5 min, 4°C). The protein concentration in the supernatant was detected by BCA detection kit (Beyotime, China). 25 μg protein per well was subjected to SDS-PAGE and then electrically transferred to PVDF membranes (Millipore, USA). The membranes were sealed with 5% skimmed milk powder for 1 h, the corresponding primary antibody [cleaved caspase 3 (1:1000, ab2302, Abcam), cleaved caspase 9 (1:1000, ab2324, Abcam), aggrecan (1:1000, ab3778, Abcam), collagen II (1:1000, ab34712, Abcam), MMP13 (1:1000, ab51072, Abcam), ADAMTS4 (1:1000, ab185722, Abcam), LC3A/LC3B (1:1000, PA1-16,930, Invitrogen), p62 (1:1000, PA5-20,839, Invitrogen), GAPDH (1:2000, ab8245, Abcam)] was added and incubated overnight at 4°C, and the secondary antibody [goat anti-rabbit (1:2000, ab150077, Abcam), goat anti-mouse (1:2000, ab150113, Abcam)] was added and incubated at room temperature for 2 h, the protein bands were developed with ECL reagent (Beyotime), and the gray value was quantified using image J 6.0 (NIH).
Bca detection kit
Manufactured by Beyotime
Sourced in China
The BCA detection kit is a laboratory reagent used to quantify the total protein concentration in a sample. It uses bicinchoninic acid (BCA) to colorimetrically detect and measure the total protein present. The kit provides the necessary solutions and standards to perform this protein quantification assay.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (13 mentions)
Ripa lysis buffer, by Beyotime (8 mentions)
Ripa reagent, by Beyotime (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab150077, by Abcam (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Gel imaging system, by GE Healthcare (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Ecl kit, by Beyotime (2 mentions)
Gapdh, by Abcam (2 mentions)
Ripa reagent, by Solarbio (2 mentions)
Anti cyclin d1, by Abcam (2 mentions)
Ecl luminescence reagent, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ecl system, by Bio-Rad (2 mentions)
Ab182858, by Abcam (2 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Ab34712, by Abcam (1 mentions)
30 protocols using bca detection kit
1
Protein Extraction and Western Blot Analysis
2
Protein Isolation and Western Blot Analysis
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Total proteins from cells or exosomes were isolated using RIPA lysis buffer (R0020; Solarbio, Beijing, China) and quantified using a BCA detection kit (P0009; Beyotime, Beijing, China) according to the manufacturer’s instructions. Proteins with suitable concentrations were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (WGPVDF22; Servicebio, Wuhai, China). The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-CD63 (ab134045; Abcam, Shanghai, China), anti-TSG101 (ab30871; Abcam, Shanghai, China), anti-VDR (PA5-109276; Invitrogen, Waltham, MA, USA), and anti-GAPDH (GB11002; Servicebio, Wuhai, China), after blocking with 5% blocking solution. Subsequently, the membranes were incubated with a secondary antibody (IH-0011; DingGuo, Beijing, China) at room temperature for 2 h. Protein bands and gray values were visualized and quantified using a gel imaging system (GE Healthcare, Chicago, IL, USA) and Image J software (NIH, Bethesda, MD, USA), respectively.
3
Western Blot Analysis of Neural Factors
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The proteins of primarily cultured NSCs or spinal cord tissues from SCI rats were isolated by RIPA lysis buffer (Beyotime, Shanghai, China), and the concentrations were determined by a BCA detection kit (Beyotime). About 20ug proteins were loaded and electrophoresed onto 12% SDS polyacrylamide gel (GE Healthcare Bioscience, Marlborough, MA, United States) and then transferred to polyvinylidene fluoride (PVDF) membrane (MilliporeSigma, Burlington, MA, United States). The membranes were blocked by 5% non-fat milk for 60 mins at 37°C and were then incubated with anti-NGF monoclonal antibody (1:1000, Abcam, Cambridge, MA, United States), anti-CREB (1:1000, Abcam), anti-GDNF (1:500, Abclonal, Wuhan, China), anti-BDNF (1:1000, Abcam), anti-VEGF (1:1000, Boster, Pleasanton, CA, United States) and anti-β-actin (1:5000, Boster) overnight at 4°C. The primary antibody incubation was followed by incubation with a secondary peroxidase-conjugated anti-rabbit (1:5000, Boster) or anti-mouse antibody (1:5000, Boster) at room temperature for 2 h. The detection of signal was conducted using Bryo ECL kit (Bytotime), and the measurement of proteins’ expression was done by Image J (Joonas “Regalis” Rikkonen, Version 1.8.0).
4
Western Blot Analysis of Cartilage Extracellular Matrix Proteins
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The total protein was extracted using RIPA reagent (Beyotime), and the protein concentration was detected using BCA detection kit (Beyotime). 40 μg protein sample/lane was subjected to 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were sealed with 5% skimmed milk at room temperature and then incubated at 4℃ overnight with the corresponding primary antibody. Following that the membranes were incubated for 2 h at room temperature with the corresponding secondary antibody. Protein bands were developed using ECL (Millipore), and images were taken and analyzed using Bio-Rad ChemiDoc XRS + (Bio-Rad, CA, USA). Primary antibodies (Anti-GJA1 (1:1000), anti-Collagen II (1:1000), anti-MMP-3 (1:2000), anti-MMP13 (1:2000), anti-ADAMTS4 (1:1000), anti-ADAMTS5 (1:1000), anti-GAPDH (1:5000)) and HRP labeled secondary antibodies (1:5000) were obtained from Abcam (Cambridge, USA). GAPDH was the normalization.
5
Western Blot Analysis of Key Signaling Proteins
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After transfection for 72 h, the total protein of cells lysed with RIPA lysis buffer (Beyotime, China) was determined by the BCA detection kit (Beyotime). Next, protein (30 µg) was isolated via 10% SDS-PAGE and transferred to PVDF membrane (Millipore, USA), and then blocked with 5% skimmed milk at 25 °C for 2 h. After removal, membranes were incubated with primary antibody: anti-p53 (ab241566; Abcam), p-STAT3 (ab76351; Abcam), STAT3 (ab68153; Abcam), p-JAK2 (ab32101; Abcam), JAK2 (ab39636; Abcam), GAPDH (ab8245; Abcam) at 4 °C overnight. After washing, the membrane was incubated with HRP-labeled secondary antibody 25 °C for 1 h, and then the signal strength of the proteins was analyzed using the ECL detection system (Millipore, USA) and Quantity One software (Bio-Rad, USA).
6
Western Blot Protein Analysis
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Cells were lysed using Radioimmunoprecipitation assay (RIPA) lysis buffer. Total proteins were extracted, and the concentrations of the proteins were analyzed using a bicinchoninic acid (BCA) detection kit (Beyotime, Shanghai, China). Protein samples were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes.
Membranes were blocked using 5% non-fat milk for 2 h and then incubated with specific primary antibodies overnight at 4 °C. After incubation with specific secondary antibodies for 2 h, the immunoreactive protein bands were visualized using the enhanced chemiluminescent (ECL) chromogenic substrate and quantified by densitometry (Quantity One; Bio-Rad, Hercules, CA, USA). Actin or Tubulin was used as a negative control.
Membranes were blocked using 5% non-fat milk for 2 h and then incubated with specific primary antibodies overnight at 4 °C. After incubation with specific secondary antibodies for 2 h, the immunoreactive protein bands were visualized using the enhanced chemiluminescent (ECL) chromogenic substrate and quantified by densitometry (Quantity One; Bio-Rad, Hercules, CA, USA). Actin or Tubulin was used as a negative control.
7
Western Blot Analysis of Apoptosis and EMT Markers
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The protein in LoVo and HCT116 cells was extracted using RIPA reagent (Solarbio), and the concentration of protein samples was measured using a BCA detection kit (Beyotime, Shanghai, China). Then sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate protein samples, and then the bands were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Subsequently, the membrane was sealed in skim milk for 4 h at 37 °C, then incubated with primary antibody for 12 h at 4 °C and secondary antibody for 2 h at 37 °C. The bands' intensities were tested using an ECL kit (Beyotime). The cleaved-caspase 3 (C-caspase 3, 1 : 500), B-cell lymphoma 2 (Bcl-2, 1 : 1000), N-cadherin (1 : 1000), E-cadherin (1 : 500), SGK1 (1 : 1000), PCNA (1 : 1000), and GAPDH (1 : 10 000) primary antibodies and goat anti-rabbit secondary antibody IgG (1 : 10 000) were obtained from Abcam (Cambridge, MA, USA).
8
Western Blot Analysis of BTG2 Protein
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Cells were lysed by cell lysis buffer (P0013, Beyotime Biotechnology, Shanghai, China), collected into a 1.5-ml EP tube, centrifuged at 12,000 rpm at 4°C for 15 min, followed by supernatant collection. The total protein concentration was determined using the BCA detection kit (P0012-1, Beyotime Biotechnology). Afterward, the proteins were added with loading buffer, boiled for 5 min, and then transferred to a PVDF membrane (Millipore, Billerica, MA, United States) through 10% SDS-PAGE gel. The membrane was sealed with 5% skimmed milk powder at room temperature for 1 h and incubated overnight with TBST diluted primary antibodies to BTG2 (ab197362; 1:1,000, Abcam) and β-actin (ab179467, 1:5,000, Abcam) at 4°C. The membrane was further incubated with the diluted horseradish (HRP)-labeled secondary goat anti-rabbit antibody (ab205718; 1:10,000, Abcam) for 1 h. Image was developed using ECL under X-ray exposure and photographed. Absorbance analysis of the bands was performed using a gel imaging analysis system. The experiment was conducted three times independently.
9
Western Blot Analysis of Cellular Proteins
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GCs were cultured according to the above methods. After washing with PBS (Solarbio, Beijing, China) precooled at 4°C, RIPA lysate (Beyotime, Shanghai, China) containing protein inhibitor was added. The supernatant was analyzed using a BCA detection kit (Beyotime). Proteins were transferred to nitrocellulose membranes by 10% SDS-PAGE (Beyotime). Then, the cells were sealed for 2 h and incubated at 4°C for 13 h with primary anti-ODC (1: 1,000) (Abcam, Shanghai, China). The cell proliferation- and apoptosis-related protein antibodies and dilution ratios were as follows: anti-PARP (1: 1,000, Beyotime), anti-β-actin (1: 2,000, TransGen Biotech, Beijing, China), anti-Cyclin D1 (1: 1,000), anti-Bcl-2 (1: 1,000) and anti-Bax (1: 1,000) (Abcam). The goat anti-rabbit IgG labeled with 1: 2,000 diluted horseradish peroxidase was incubated at room temperature for 1 h and then washed with TBST (Beyotime). Finally, enhanced chemiluminescence reagent (Beyotime) was used for development on a gel imaging system instrument. Image Lab (Bio-Rad, Shanghai, China) was used to analyze the optical density levels and to calculate the relative protein content.
10
Western Blot Analysis of Proteins
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Total protein of tissues or cells (PANC-1 and SW1990) was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein content was detected using BCA detection kit (Beyotime Institute of Biotechnology). A total of 30 µg protein sample/well was separated using 10% SDS-PAGE and then transferred to PVDF membranes (MilliporeSigma). Following that, the membranes were incubated for 1 h with 5% skimmed milk powder at room temperature. The membranes were incubated overnight at 4˚C with the corresponding primary antibodies: SIRT6 (1:1,000; catalog no. ab119007), NF-κB p65 (1:1,000; catalog no. ab32536), lamin B (1:1,000; catalog no. ab232731), IκBα (1:1,000; catalog no. ab76429), GPX4 (1:1,000; catalog no. ab125066), SLC7A11 (1:1,000; catalog no. ab37185), HK2 (1:1,000; catalog no. ab104836) and lactate dehydrogenase A (LDHA; 1:1,000; catalog no. ab134187) (all from Abcam). Following the primary incubation, membranes were washed three times with PBST (0.1% Tween20) and then incubated at room temperature for 2 h with the DyLight® 488-conjugated secondary antibodies: Goat anti-rabbit IgG H&L (1:5,000; product code ab96899) or goat anti-mouse IgG H&L (1:5,000; product code ab96879; both from Abcam). Protein bands were visualized using Immobilon ECL Ultra Western HRP (MilliporeSigma). The results were analyzed using ImageJ version 6.0 (National Institutes of Health).
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