First, the protein of testicular samples in different groups were extracted using RIPA lysis buffer (P0013B, Beyotime, Shanghai) and protease inhibitor (P1005, Beyotime, Shanghai) via homogenate. Besides, BCA protein assay kit (P0009, Beyotime, Shanghai) was applied to determine the protein concentration. Protein samples (10-30 µg protein per lane) were electrophoresed using the 10 % SDS-PAGE gel and wet transferred applying the PVDF membrane (ISEQ00010, Millipore, USA). After blocking via 5 % skimmed milk for 2 h at room temperature, the transferred PVDF membranes were incubated with LC3A/B (D3U4C) (1:1000 dilution) rabbit monoclonal antibody (12741S, Cell Signaling Technology, USA), SQSTM1/p62 (1:1000 dilution) rabbit polyclonal antibody (5114S, Cell Signaling Technology, USA), LAMP1 (1:1000 dilution) polyclonal monoclonal antibody (A2582, ABclonal, P.R.China), PINK1 (1:1000 dilution) rabbit monoclonal antibody (ab216144, Abcam, USA), Parkin (1:1000 dilution) mouse monoclonal antibody (ab77924, Abcam, USA) and β-Actin Rabbit monoclonal antibody (AC026, ABclonal, P.R.China) at 4°C overnight. After incubation via goat anti-rabbit IgG (1:5000, HS001-01, Transgen, Beijing) for 2 h, the results were chemical developed using automatic chemiluminescence image analysis system (4600, Tanon, Shanghai). The quantitative analysis of western blot results was analysed using Image J.
Bca protein assay kit
Manufactured by Beyotime
11 060 citations
Sourced in China, United States, Germany, Japan, Puerto Rico, United Kingdom, Switzerland, Sweden
About the product
The BCA protein assay kit is a colorimetric-based method for the quantitative determination of total protein concentration in a sample. It uses bicinchoninic acid (BCA) to detect and quantify the presence of protein.
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Market Availability & Pricing
The BCA Protein Assay Kit is commercialized by Beyotime Biotechnology and available through authorized distributors. Prices typically range from RMB 150.00 to RMB 1,398.00, depending on the package size.
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«Bca protein assay kit» FAQ
11 060 protocols using «bca protein assay kit»
1
Testicular Protein Extraction and Western Blot
2
Quantifying c-di-GMP in Vibrio parahaemolyticus
The intracellular c-di-GMP concentration was measured as previously described [26 (link)]. The V. parahaemolyticus suspensions were co-cultured with hexanal (0, 1/8 MIC, and 1/4 MIC) at 37 °C for 48 h. After centrifugation and washing, the V. parahaemolyticus cells were suspended in ice-cold saline and incubated at 100 °C for 5 min. The samples were sonicated (100 w, on/2 s, off/2 s) in an ice-water bath for 6 min and centrifuged at 4 °C, 8000× g for 5 min. The supernatant was collected, and the cell sediments were sonicated another two times to fully extract the intracellular c-di-GMP. The total protein and c-di-GMP levels in the supernatant were determined using the BCA protein assay kit and the Enzyme-Linked Immunosorbent Assay kit (Beyotime, Beijing, China), respectively. Finally, the intracellular c-di-GMP concentration was expressed as pmol/mg protein.
3
Polyamine Metabolism Analysis Protocol
Sodium hydroxide (NaOH), sodium bicarbonate (NaHCO3), ammonia (analytical reagent), perchloric acid (HClO4; analytical reagent), chloroform (CHCl3; analytical reagent) and isopropyl alcohol were purchased from Damao chemical reagent factory. Acetonitrile (C2H3N; HPLC) was purchased from Tianjin Kemiou chemical reagent Co.,Ltd. Dansyl chloride (DNSCl) was purchased from Shanghai Macklin biochemical technology Co., Ltd. Spermine (standard), spermidine (standard) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Spermidine/Spermine N1-Actyltransferase (SSAT) ELISA kit, hypoxia-inducible factor-1α (HIF-1α) ELISA kit and nitric oxide synthase (NOS) ELISA Kit were purchased from Quanzhou Ruixin biotechnology Co.,Ltd. PBMCs isolation medium was purchased from Tianjin Haoyang biological manufacture Co., Ltd. Spermine oxidase (SMOX) antibody, ornithine decarboxylase 1(ODC1) antibody, spermidine synthase (SRM) antibody and GAPDH antibody were purchased from Proteintech Group. RIPA lysis buffer, Proteinase K, BCA protein assay kit, enhanced chemiluminescence (ECL) detection reagent, HRP-labeled goat anti-mouse IgG and HRP-labeled goat anti-rabbit IgG were purchased from Beyotime biotechnology. TRIzol was purchased from Thermo Fisher Scientific. Evo M-MLV reverse transcription kit was purchased from Hunan Accurate biotechnology. TB Green® Premix Ex Taq™ was purchased from Takara.
4
Western Blot Protein Analysis
HOS and MG63 cells were treated with RIPA buffer (Beyotime) to extract proteins, and the protein concentration was quantified using the BCA Protein Assay Kit (Beyotime). A total of 20 µg of protein per well was loaded onto a 10% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore). The membrane was then incubated overnight at 4 °C with the respective primary antibodies (1:500, Invitrogen, USA, PA5-41224), followed by a 2-hour incubation with secondary antibodies at room temperature. Protein bands were visualized using a chemiluminescence ECL kit (Thermo Fisher Scientific), with GAPDH utilized as the loading control.
5
Quantification and Western Blotting Protocol
The proteins were quantified by the BCA Protein Assay Kit (Beyotime, Shanghai, China). The Western blotting was performed according to the standard protocol using GBP5 antibody (1:2000, Proteintech, China) and GAPDH antibody (1: 10000, Protintech, China) and blots was detected using the ECL assay kit (Thermo Scientific, Waltham, MA, USA).
Top 5 most cited protocols using «bca protein assay kit»
1
Western Blot Analysis of Protein Expression
The RIPA kit (Beyotime, Shanghai, China) supplemented with phenylmethanesulfonyl fluoride was used to extract protein lysate from CRC tissues and cells according to the protocols. The BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to quantify the protein concentration. The protein lysate was separated via 10% SDS-PAGE (Beyotime, Shanghai, China), next, it was transferred onto a polyvinylidene fluoride membrane (Millipore, USA). The membranes were blocked with 5% skim milk powder for 2 h and then incubated with the specific primary antibody overnight at 4°C, followed by a wash in TBST. Then, the membrane was incubated with the corresponding secondary antibody for 2 h at room temperature and then rinsed in TBST for three times (10 min each time). The level of protein expression was detected by ECL Plus (Millipore, USA) using a Bio-Imaging System (Bio-Rad, USA). The antibodies against SP1, IκBα, p- IκBα, p65, p-p65, E-cadherin, ZO-1, N-cadherin, Vimentin, HIF-1α, Histone H3, and β-actin were purchased from Abcam (Cambridge, MA, USA).
Corresponding organizations : Jiangsu Province Hospital, Nanjing Medical University
2
Western Blot Analysis of Protein Expression
After washing twice with cold PBS, cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) with 1 mM PMSF and protease inhibitor cocktail on ice for 30 min, manually scraped from culture plates and then quantified using the BCA Protein Assay Kit (Beyotime). Proteins were separated on 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, electroblotted onto a polyvinylidene difluoride (PVDF) membrane (0.22 μm, Millipore, Billerica, MA, USA). The membranes were blocked with 5 % BSA and incubated with specific antibodies overnight at 4 °C and then were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The primary antibodies were as follows: IKKα/β, phosphorus IKKα/β, p65, phosphorus p65, JNK, phosphorus JNK, phosphorus p38 (1/1000, Cell Signaling Technology, USA), CD63, HSP70 (1/1000, Abcam), GAPDH (1/1000, Santa cruz), and β-actin (1/1000, Zhongshan, Beijing China). Secondary (HRP)-conjugated antibodies were purchased from NeoBioscience. Antibody and antigen complexes were detected using chemiluminescent ECL reagent (Millipore, USA).
Corresponding organizations : Chinese Academy of Medical Sciences & Peking Union Medical College, Peking Union Medical College Hospital
3
Quantitative Western Blot Analysis of BBB Proteins
Challenged hBMEC and mice brains were lysed in RIPA buffer with protease inhibitor cocktail (Sigma-Aldrich, USA), sonicated and centrifuged at 10,000 g for 10 min at 4°C. The insoluble debris was removed and protein concentration in supernatant was measured using BCA protein assay kit (Beyotime, China). Brain extracts or cell lysates were then separated on 10%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, CA). The blots were blocked in 5% BSA in Tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature, and then incubated overnight with either one of anti-ZO-1 (Abcam, USA), Claudin-5 (AVIVA, USA), Occludin (AVIVA, USA), β-Catenin (HuaAn, China), β-actin (HuaAn, China), ERK1/2 (CST, USA), p-ERK1/2 (CST, USA) antibodies. After washing, the blots were incubated with species-specific horseradish peroxidase-conjugated antibodies and finally visualized with the ECL reagents (Bio-rad, USA). All Western Blots were densitometrically quantified by using the ImageJ software, and the results were analyzed as the relative immunoreactivity of each protein normalized to the respective loading control.
Corresponding organizations : Huazhong Agricultural University
4
Protein Expression Analysis in Glioma Cells
LN-229, U-87 and A-172 glioma cell lines were harvested, washed with ice-cold PBS buffer and then suspended in RIPA lysis buffer (Beyotime, China) with phosphatase inhibitors (Sigma Aldrich, St. Louis, MO, USA) and complete protease inhibitor cocktail (Roche). Protein concentrations were detected with a BCA protein assay kit (Beyotime Biotech, China). Then, 30 μg of lysate was subjected to 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, USA). After being blocked with 5% bovine serum albumin (BSA) in TBST buffer at room temperature for 1–4 h, the PVDF membranes were gently incubated with specific primary antibodies against human tubulin (1:1000), CDK2 (1:1000), CDK6 (1:1000), cyclin D1 (1:1000), GFAP (1:1000), β3-tubulin (1:1000), E-cadherin (1:1 000), vimentin (1:1000), CDC6 (1: 10,000), CDT1 (1:10,000), CDC45 (1:10,000), MCM2 (1:1000), MCM3 (1:10,000), ORC6 (1:1 000), MYBL2 (1:100) and Flag tag (1:1000) at 4 °C overnight. After a wash with TBST buffer, the PVDF membranes were incubated with horseradish peroxidase (HRP)-labelled goat anti-mouse IgG (H + L) (A0216, 1:10,000) or goat anti-rabbit IgG (H + L) (A0208, 1:10,000) at room temperature for 2 h. Protein bands were visualised with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher, Waltham, MA, USA), and luminescence images were analysed with a Western blot detection instrument (Clinx Science, Shanghai, China).
Corresponding organizations : Southwest University
5
Western Blot Analysis of Protein Expression
Total protein was extracted using RIPA solution, and quantified with BCA protein assay kit (Beyotime, Shanghai, China). Protein samples (20 µg) were then subjected to electrophoresis on SDS-PAGE gels (Solarbio, Beijing, China) before transferring onto PVDF membranes (Millipore, USA). After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China). Then, the PVDF membranes were washed with TBST, followed by incubation for 1 h with anti-rabbit or anti-mouse IgG-peroxidase secondary antibody (1: 3000) at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) system and analyzed by Gel-Pro-Analyzer software.
Corresponding organizations : China Medical University
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