Cells were washed with Dulbecco's phosphate-buffered saline (DPBS, Gibco BRL, Life Technologies) and proteins were extracted using mammalian cell lysate buffer (Biyuntian, Beijing, China). Samples were then centrifuged at 14000 rpm for 5 min at 4°C and protein concentrations of the supernatants were determined by the bicinchoninic acid (BCA) protein concentration detection kit (Biyuntian, Beijing, China). The extracted proteins were separated by 12% SDS-PAGE, transferred onto a 0.22 μM nitrocellulose membranes, and incubated with mouse anti-α-actin (TA-09, ZSGB-BIO, Beijing, China) or mouse anti-LL-37 (sc-166770, Santa Cruz Biotechnolology). Membranes were washed and then incubated with HRP-conjugated goat anti-mouse antibodies (EM35110, EMAR, Beijing, China). Immunoreactions were visualized using Pierce Western ECL substrate (Thermo Scientific, Rockford, IL) or Clarity™ Western ECL Substrate (BioRad, Hercules, CA) and exposed to Amersham Hyperfilm ECL film (GE Healthcare Biosciences, Piscataway, NJ). Protein bands were quantified by Quantity One analysis (BioRad).
Bca protein concentration detection kit
Manufactured by Beyotime
Sourced in China
The BCA protein concentration detection kit is a laboratory equipment that provides a colorimetric detection method for determining the total protein concentration in a sample. The kit utilizes the bicinchoninic acid (BCA) reaction to measure the protein content, with the resulting color change being proportional to the protein concentration present.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Ripa lysate, by Beyotime (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Pierce ecl western substrate, by Thermo Fisher Scientific (1 mentions)
Amersham hyperfilm ecl film, by GE Healthcare (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Beyoecl plus, by Beyotime (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ovcar3, by American Type Culture Collection (1 mentions)
Iose80, by American Type Culture Collection (1 mentions)
Dual luciferase report detection system, by Promega (1 mentions)
Lipofectamine tm 2000 transfection kit, by Thermo Fisher Scientific (1 mentions)
Ecl chemiluminescence reagent, by Beyotime (1 mentions)
Mirna extraction kit, by Thermo Fisher Scientific (1 mentions)
Deme medium, by Thermo Fisher Scientific (1 mentions)
Image lab version 3, by Bio-Rad (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
26 protocols using bca protein concentration detection kit
1
Protein Extraction and Western Blot Analysis
2
Western Blot Protein Detection
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Cells were lysed in RIPA buffer (strong) (biyuntian, China). The concentration of total proteins was determined using BCA protein concentration detection kit (biyuntian, China). The same amount of proteins were loaded on SDS-PAGE and resolved by electrophoresis, followed by transferring onto NC membrane. Then, the membranes were blocked with blocking buffer (5% fat-free dry milk in TBST buffer) and incubated with primary antibodies and HRP labeled secondary antibodies, respectively. Lastly, specific proteins were visualized with enhanced ECL plus Western blotting substrate according the manufacturer’s instructions (Thermo).
3
Western Blot Analysis of Protein Expression
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Firstly, radioimmunoprecipitation assay buffer (cat. no. P0013B; Shanghai Biyuntian Biotechnology Co., Ltd.) was added to the tissue or cells to extract proteins, and the protein concentration was measured using a BCA protein concentration detection kit (cat. no. P0010; Shanghai Biyuntian Biotechnology Co., Ltd.). Subsequently, protein loading buffer was added (cat. no. P0015; Shanghai Biyuntian Biotechnology Co., Ltd.) and lysates were heated at 95°C for 10 min to denature the protein. In turn, liver tissue lysates or cell lysates (~20 µg) were subjected to SDS-PAGE (10%). Subsequently, the protein was transferred to the PVDF membrane at 4°C and PVDF membrane was soaked in QuickBlock™ Blocking Buffer (cat. no. P0220; Shanghai Biyuntian Biotechnology Co., Ltd.) at room temperature for 15 min. The membrane was incubated at 4°C for 14 h with the following primary antibodies against: PTEN (1:1,000), AKT (1:1,000), p-AKT (1:1,000), IκBα (1:1,000), p-p65 (1:1,000), t-p65 (1:1,000) and GAPDH (1:5,000). Following the primary incubation, the membrane was incubated with secondary antibody (1:10,000) at room temperature for 1 h. Subsequently, protein visualization was performed using BeyoECL Plus (Beyotime Institute of Biotechnology) and Image Lab 2.5.2 software (Bio Rad Laboratories, Inc.). GAPDH was used as an internal reference protein.
4
Ovarian Cancer Cell Line Characterization
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Human ovarian cancer epithelial cell line OVCAR3 and human normal ovarian epithelial cell line IOSE80 were purchased from ATCC cell bank in the United States. Cell culture reagents (DEME medium, fetal bovine serum, streptomycin penicillin, trypsin) were purchased from Gibco, USA. Cell proliferation activity assay Kit CCK-8 was purchased from Tongren Institute of Chemistry, Japan. miRNA extraction kit, miRNA reverse transcription and fluorescence quantitative kit, and Lipofectamine TM 2000 transfection kit were all purchased from Invitrogen, USA. Mir-30b-3p, mimics and mimic control miRNAs were synthesized by Shanghai Gemar Pharmaceutical Technology Co., LTD., China. Mir-30b-3p and U6 primers were designed and synthesized by bioengineering (Shanghai) Co., LTD., China. ECL chemiluminescence reagent and BCA protein concentration detection kit were purchased from Shanghai Biyuntian Biotechnology Co., LTD., China. The dual luciferase report detection system is the product of Promega, USA.
5
Western Blot Analysis of NOS2 Protein
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After extraction of proteins using RIPA buffer (Beyotime Institute of Biotechnology), BCA protein concentration detection kit (cat. no. P0009; Beyotime Institute of Biotechnology) was used to measure the concentrations of target proteins. The protein samples were mixed with SDS-PAGE loading buffer before boiling for 5 min. Then, 20 µg protein samples were loaded for 10% SDS-PAGE. On ice bath, the samples were transferred onto PVDF membrane at 100 V for 2 h, and then blocked with 5% skimmed milk at room temperature for 1 h. Then, primary antibodies of NOS2 (cat. no. ab3523; 1:800; rabbit anti-rat, polyclonal; Abcam) and internal reference β-actin (cat. no. ab129348; 1:5,000; rabbit anti-rat, Abcam) were used to incubate the membrane at 4˚C overnight. Afterwards, secondary antibody (cat. no. ab6721; 1:3,000; goat anti-rabbit; Abcam) was used to incubate the membrane at room temperature for 1 h. The membrane was then soaked in electrochemiluminescence liquid (cat. no. ab65623; Abcam), and imaged in gel imaging system. Then, Image Lab (version 3.0; Bio-Rad Laboratories, Inc.) was used to analyze the protein bands, and the relative expression of target protein was expressed as the ratio of target protein greyscale against β-actin greyscale.
6
Protein Expression Analysis in Myocardium
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The total protein of myocardial tissue and cardiomyocyte was extracted by RIPA lysate (Beyotime), and BCA protein concentration detection kit (Beyotime) was used for the measurement of protein concentration. The same amount of total protein was separated in SDS-PAGE (12 %), and then transferred to PVDF membrane by electroporation. After washed for 3 times, the membranes were sealed for 1 h at room temperature with 5 % skimmed milk powder. After washed for 3 times, the membranes were incubated overnight at 4 °C with appropriate primary antibody. After washed for 3 times, the membranes were incubated for 2 h at room temperature with corresponding secondary antibody. After washed for 3 times, the bands were detected. Used antibodies were as follow: anti-Bcl-2 (ab196495, 1:1000), anti-Bax (ab53154, 1:1000), anti-IGF2R (ab124767, 1:1000), anti-β-actin (ab8226, 1:1000) and anti-GAPDH (ab8245, 1:1000). All antibodies were acquired from Abcam (Cambridge, USA). GAPDH or β-actin was used as normalizations.
7
Western Blot Analysis of LncRNA HOXA-AS2, miR-885-5p, and KDM5B in PCa
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Total protein was extracted from the cells using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China), and the protein concentration was determined using a BCA protein concentration detection kit (Beyotime, China). Then, 30 μg of proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel LncRNA HOXA-AS2, miR-885-5p, and KDM5B in PCa electrophoresis, transferred to polyvinylidene difluoride membranes, and blocked with 5% skim milk. Next, the membranes were incubated with primary antibodies against KDM5B (1:1,000, Abcam, USA) and GAPDH (1:1,000, Abcam, USA) at 4°C for 12 h. After incubation with the primary antibodies, the membranes were incubated with an HRP-conjugated secondary antibody (1:5,000, Abcam, USA) for 3 h at 37°C. The protein bands in the membranes were visualized using an ECL reagent (Sigma-Aldrich, USA).
8
Western Blot Analysis of EMT Markers
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Tissues or cells were lysed utilizing RIPA lysate (Beyotime) on the ice. The sample was centrifuged at 12,000 g for 20 min at 4 °C. The supernatant was collected and stored at -80 °C. Protein concentration was examined via BCA protein concentration detection kit (Beyotime). Protein sample was separated via SDS-PAGE and transferred into PVDF membrane (Bio-Rad, Hercules, CA). 5% non-fat soluble milk liquid was utilized to block the membrane for 1 h. Afterwards, the membrane was incubated with primary antibodies including E-cadherin (1:1000, ab133597, abcam, Cambridge Science Park, UK), N-cadherin (1:1000, ab207608), Vimentin (1:1000, ab92547) and GAPDH (1:1000, ab9485) at 4 °C overnight, followed by goat anti-rabbit IgG H&L (HRP; 1:5000, ab205718, abcam) at room temperature for 1 h. Protein blots were visualized utilizing ECL chemiluminescence reagent (Pierce, Rockford, USA). The grayscale value was measured using the ImageJ software.
9
Western Blot Analysis of Protein Expression
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Kidney tissues were homogenized and lysed in RIPA lysis buffer (Bioteke Corporation, China). Cells were lysed in RIPA lysis buffer directly. After centrifugation, supernatants were collected and protein concentrations were detected using a BCA protein concentration detection kit (Beyotime, China). Equal amounts of protein were separated by 7.5–12.5% SDS-polyacrylamide gel electrophoresis (PAGE) (Beyotime, China) and transferred onto PVDF membranes. The membranes were blocked with 2% BSA for 1 h at room temperature and incubated with primary antibodies at 4°C overnight. The primary antibodies used were as follows: anti-SRC (phosphor Y529) antibodies (Abcam, 1 : 10000), mouse B7-1/CD80 affinity purified polyclonal antibodies (R&D system, 1 μg/ml), and anti-GAPDH polyclonal antibodies (Proteintech, 1 : 20000). Blots were then washed and incubated with peroxidase-conjugated affinipure goat anti-rabbit or rabbit anti-goat secondary antibodies (IgG-HRP; ZSGB-BIO, Beijing, China, 1 : 5000) at room temperature for 1 h. After several washes, visualization was performed on an ECL Western blotting detection system (GE healthcare; RPN2106).
10
Western Blot Analysis of Protein Expression
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After FLO treatment, cells were washed with cold PBS and lysed using RIPA lysis buffer (Solarbio, Beijing, China). The whole cell lysates were prepared for Western blot analysis. Protein concentrations were determined using the BCA protein concentration detection kit (Beyotime, Beijing, China). Equal amounts of protein (30 μg) were separated using SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with a PBST solution containing 5% skim milk for 2 h.
Next, the membranes were incubated with the primary antibodies at 4 °C overnight. They were then washed six times for 5 min each with PBST and subsequently incubated with an IgG horseradish peroxidase-labeled secondary antibody (ZSGB-BIO, Beijing, China). After another six washes of 5 min each with PBST, the membranes were visualized using super-sensitivity chemiluminescence reagents (Willget Biotech, Shanghai, China).
Next, the membranes were incubated with the primary antibodies at 4 °C overnight. They were then washed six times for 5 min each with PBST and subsequently incubated with an IgG horseradish peroxidase-labeled secondary antibody (ZSGB-BIO, Beijing, China). After another six washes of 5 min each with PBST, the membranes were visualized using super-sensitivity chemiluminescence reagents (Willget Biotech, Shanghai, China).
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