The protein was contained by lysing tissues and cells into RIPA buffer (Beyotime), electrophoresed on SDS-PAGE (Solarbio, Beijing, China) and then blotted onto PVDF membranes (Millipore, Billerica, MA, USA). After blockage in 5% skim milk for 1 h, the proteins were interacted overnight with primary antibodies and secondary antibody (bs-0295M-HRP; Bioss, Beijing, China) for 1.5 h. The bands were visualized with ECL reagent (Beyotime) and band intensity was examined with ImageJ (NIH, Bethesda, MD, USA). The primary antibodies including β-actin (bs-0061R; Bioss), hexokinase2 (HK-2; bs-3993R; Bioss), CyclinD1 (bs-0623R; Bioss), MMP9 (bs-4593R; Bioss), or KLF12 (bs-16783R; Bioss)
Bs 0295m hrp
Manufactured by Bioss Antibodies
Sourced in China
The Bs-0295M-HRP is a horseradish peroxidase (HRP) conjugated secondary antibody produced by Bioss Antibodies. It is designed for use in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry, where it can be used to detect and visualize target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (4 mentions)
Sodium dodecyl sulfonate polyacrylamide gel, by Solarbio (4 mentions)
Bca protein quantification kit, by Vazyme (3 mentions)
Bs 2188r, by Bioss Antibodies (3 mentions)
β actin bs 0061r, by Bioss Antibodies (2 mentions)
Polyvinylidene difluoride membrane, by Cytiva (2 mentions)
Ripa buffer, by CWBIO (2 mentions)
Ecl kit, by Beyotime (2 mentions)
Sds page, by Beyotime (2 mentions)
Bs 0061r, by Bioss Antibodies (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Sds page, by Solarbio (1 mentions)
Mmp9 bs 4593r, by Bioss Antibodies (1 mentions)
Chemiluminescent substrate kit, by Merck Group (1 mentions)
Ecl reagent, by Vazyme (1 mentions)
Bs 10009r, by Bioss Antibodies (1 mentions)
Bs 0623r, by Bioss Antibodies (1 mentions)
Bca protein assay kit, by Tiangen Biotech (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
10 protocols using bs 0295m hrp
1
Western Blot Protein Analysis
2
Protein Quantification and Western Blot
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The isolation of total protein was executed utilizing RIPA buffer (CWBio, Beijing, China) and the concentrations of proteins were measured with a BCA Protein Quantification Kit (Vazyme, Nanjing, China). Then equal amounts of protein were separated through sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) electrophoresis followed by transferring into polyvinylidene difluoride membranes (Amersham Biosciences, Chicago, IL, USA). After blocking in 5% non-fat milk for 1 h at indoor temperature, the membranes were incubated overnight with primary antibodies: β-actin (bs-0061R; Bioss, Beijing, China) and HMGB1 (bs-0664R; Bioss) at 4°C and probed with horseradish peroxidase-conjugated secondary antibody (bs-0295M-HRP; Bioss) for 1 h at indoor temperature. The signals were examined using chemiluminescent substrate kit (Millipore, Bedford, MA, USA).
3
Protein Quantification and Western Blot Analysis
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Total protein in GC cells was extracted utilizing RIPA buffer (Beyotime) and determined utilizing a BCA protein assay kit (Tiangen, Beijing, China). Then the equal amount of protein (30 μg) was separated by sodium dodecyl sulfonate–polyacrylamide gel (Solarbio) and blotted onto polyvinylidenedifluoride membranes (Amersham Biosciences, Chicago, IL, USA). After blocking in 5% defatted milk for 1 h, the membranes were cultivated overnight with primary antibodies against CyclinD1 (1:2,000; bs-0623R; Bioss, Beijing, China), E-cadherin (1:2,000; bs-10009R; Bioss), N-cadherin (1:2,000; bs-1172R; Bioss), or GAPDH (1:5,000; bs-2188R; Bioss) at 4°C followed by interaction with HRP-conjugated secondary antibody (1:5,000; bs-0295M-HRP; Bioss) for 1.5 h at room temperature. The protein bands were visualized with an ECL reagent (Vazyme, Nanjing, China) and analyzed via ImageJ v1.8.0 (National Institutes of Health).
4
Protein Expression Analysis of Epithelial-Mesenchymal Transition
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The tissues and cells were subjected to RIPA (Beyotime, Shanghai, China) for total protein extraction. The proteins were then separated using SDS-PAGE (Beyotime) and blotted onto PVDF membranes (Beyotime). The membranes were then blocked with 5 % slim milk, incubated with primary antibodies, followed by interaction with a secondary antibody (bs-0295 M-HRP; Bioss, Beijing, China). The proteins were detected with an ECL kit (Beyotime). The primary antibody included anti-ONECUT2 (bs-19643R; Bioss), anti-Zinc Finger E-Box Binding Homeobox 2 (anti-ZEB2; bs-20484R; Bioss), anti-E-cadherin (bs-1519R; Bioss), anti-Vimentin (bs-0756R; Bioss), anti-vascular endothelial growth factor A (VEGFA) (bs-20393R; Bioss) and β-actin (bs-0061R; Bioss).
5
Immunohistochemical Analysis of CD34 Expression
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Tumor paraffin sections were successively dewaxed and hydrated, antigen repair, serum blocking, CD34 antibody (1 : 2500, ab81289; Abcam, UK) staining-secondary antibody (BS-0295M-HRP; (Bioss, China) incubation-DAB coloration-gradient alcohol dehydration-neutral gum sealing sheet. The protein expression was observed under a microscope.
6
Protein Extraction and Western Blot Analysis
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Protein extraction was finished using RIPA buffer (CWBio, Beijing, China). The protein concentrations were examined via a BCA Protein Quantification Kit (Vazyme). Then, an equal amount of proteins was separated through sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) electrophoresis and blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk for 1 h at indoor temperature and immunoblotted with primary antibodies against hexokinase2 (HK2; bs-9455R; Bioss, Beijing, China), CPEB4 (bs-14020R; Bioss) and β-actin (bs-0061R; Bioss) overnight at 4°C followed by interaction with indicated horseradish peroxidase-conjugated secondary antibody (bs-0295M-HRP; Bioss) for 1 h. The immunoreactive bands were developed using ECL kit (Beyotime, Shanghai, China) and analyzed using Image J software.
7
Protein Extraction and Western Blot Analysis
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Protein extraction was finished using RIPA buffer (Beyotime) and protein concentration determination was executed utilizing a BCA Protein Quantification Kit (Vazyme). 20 μg proteins were separated by 10% sodium dodecyl sulfonate-polyacrylamide gel (Solarbio, Beijing, China) electrophoresis. Next, the proteins were blotted onto polyvinylidene difluoride membranes (Pall Corporation, New York, NYC, USA). Afterward, the membranes were blocked utilizing 5% nonfat milk for 1 h at room temperature and incubated with primary antibodies against GAPDH (bs-2188R; 1:5,000; Bioss, Beijing, China), B-cell lymphoma-2 (Bcl-2; 1:2,000; bs-34012R; Bioss), BCL2-Associated X (Bax; 1:2,000; bs-0127R; Bioss), cleaved caspase-3 (bs-0081R; 1:2,000; Bioss), and PDE7B (bs-11576R; 1:2,000; Bioss) overnight at 4°C and secondary antibody (bs-0295M-HRP; 1:5,000; Bioss) for 1 h at room temperature, followed by ECL detection.
8
Immunohistochemical Analysis of Ki-67 Expression
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The tumor tissues obtained from the nude mice were embedded in paraffin and cut into sections for immunohistochemistry analysis. After deparaffinization, rehydration, antigenic retrieval, blocking in goat serum, the sections were incubated with primary Ki-67 antibody (1:100, bs-23103R, Bioss) at 4°C overnight and developed with mouse anti-rabbit IgG/HRP (1:100, bs- 0295M-HRP, Bioss). The sections were subjected to counterstaining with hematoxylin, and detected under a microscopy. The number of Ki-67 positive cells was counted.
9
Western Blot Analysis of E2F7, VEGFR2, and Phospho-VEGFR2
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Cellular proteins were extracted with RIPA lysis buffer (Sigma Aldrich), separated using SDS-PAGE (Beyotime), and transferred to polyvinylidene fluoride membranes (Beyotime). Membranes were then blocked in 5% skim milk and treated with anti-E2F7 (1 : 1000, ab56022; Abcam, UK), VEGFR2 (1 : 300, bs-10412R; Bioss, China), p-VEGFR2 (1 : 1000, ab5473; Abcam, UK), GAPDH (1 : 3000, bs-2188R; Bioss, China), and mouse anti-rabbit secondary antibody (1 : 5000, bs-0295M-HRP; Bioss, China). Western blots were visualized using the ECL system (Beyotime).
10
Protein Expression Analysis Protocol
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RIPA buffer (Beyotime) was used to extract total proteins from cells and tissues. The protein concentration of samples was determined by BCA (Beyotime) method. The proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then the gels were transferred onto the PVDF (Millipore, Billerica, MA, USA) membrane and sealed with 5% milk for 1 h. PVDF membranes were incubated with the primary antibody against proliferating cell nuclear antigen (PCNA; ab18197, 1: 1000, ABCAm), Bax (ab32503; 1:1000; ABCAm), E-cadherin (ABCAm, ab40772, 1:1000) and β-actin (bs-0061R; Bioss, Beijing, China) overnight at 4°C. Then, secondary antibody (bs-0295M-HRP; Bioss) were incubated to membranes for 1 h. ECL (Beyotime) hypersensitive luminescence solution was added to visualize the blots, and Image Lab was used to analyze the gray value of the blots
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