Total protein was isolated from differently treated cells using RIPA lysis buffer (Beyotime Institute of Biotechnology), and the total protein concentration was measured using a BCA protein assay kit. Subsequently, the protein samples (20 µg/lane) were separated using 10% SDS-PAGE, transferred to PVDF membranes and blocked with 5% skimmed milk. After blocking at 37°C for 1 h, the membranes were incubated with anti-β-catenin (catalogue number, 17565-1-AP; 1:1,000), anti-c-Myc (catalogue number, 10828-1-AP; 1:1,000), anti-cyclin D1 (catalogue number, 26939-1-AP; 1:1,000) and anti-GAPDH (catalogue number, 10494-1-AP; 1:2,000) antibodies, all from ProteinTech Group, Inc., or the aforementioned anti-HSP70, anti-TSG101, anti-CD9 and anti-calnexin antibodies at 4°C overnight. After washing, the membranes were incubated with goat anti-rabbit/mouse IgG (H+L)-HRP secondary antibody (catalogue number, 111-035-003/115-035-003; 1:5,000; Jackson ImmunoResearch Laboratories, Inc.) at 37°C for 2 h. After washing with PBST (1,000 ml 1X PBS + 1 ml Tween-20) five times, the protein bands were visualized using a Millipore ECL system (Tanon Science & Technology Co., Ltd.). The protein bands were analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics Imaging Technologies Inc.).
Anti c myc
Manufactured by Proteintech
Sourced in China, United States
The Anti-c-Myc antibody is a laboratory tool used for the detection and analysis of the c-Myc protein in various cellular and molecular biology applications. It is a specific antibody that binds to the c-Myc protein, allowing researchers to identify and quantify its presence in samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Proteintech (10 mentions)
Anti cyclin d1, by Proteintech (9 mentions)
Anti β actin, by Proteintech (9 mentions)
Anti β catenin, by Proteintech (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
Anti beta catenin, by Proteintech (3 mentions)
Anti e cadherin, by Proteintech (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Anti p21, by Proteintech (2 mentions)
Anti c jun, by Bioworld Technology (2 mentions)
Anti mmp1, by Proteintech (2 mentions)
Anti tcf4, by Cell Signaling Technology (2 mentions)
Anti bcl 2, by Abcam (2 mentions)
Anti p akt ser473, by Proteintech (2 mentions)
Amersham imager 680, by GE Healthcare (2 mentions)
Ecl western blotting protocol, by GE Healthcare (2 mentions)
Anti p pi3k tyr458, by Cell Signaling Technology (2 mentions)
Anti akt, by Proteintech (2 mentions)
Anti flag, by Merck Group (2 mentions)
41 protocols using anti c myc
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis Protocol
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Cell and tissue proteins were extracted using RIPA (Beyotime Biotechnology). Protein concentration was detected by the BCA kit and the protein loading was 10–40 µg. Proteins were separated by using a 10% gel for polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes and blocked for 2 h with 5% skimmed milk at room temperature. Then, proteins on the membrane were detected with primary antibodies for 16–18 h at 4°C, including anti-β-catenin (1:1,000; cat. no. bsm-33194M; BIOSS), anti-NDRG3 (1:750; cat. no. BS62436; Bioworld Technology, Inc.), anti-c-Myc (1:6,000; ca. to. 10828-1-AP; Proteintech Group, Inc.), anti-MDR1 (1:1,000; ca. to. 13342S; Cell Signaling Technology), anti-LaminB1 (1:6,000; cat.no.12987-I-AP; Proteintech Group, Inc.) and anti-GAPDH (1:6,000; cat. no. BS65483M; Bioworld Technology, Inc.). After 2 h of incubation with goat anti-rabbit IgG H&L HRP conjugate secondary antibody (1:6,000; cat. no. BS13278; Bioworld Technology, Inc.) at 4°C, protein bands were visualized using BeyoECL Plus (Beyotime Institute of Biotechnology). Finally, the densitometric analysis of the protein was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Immunophenotyping of Cultured Cells
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IHC analysis was performed on the cell-block sections from the cultured cells by using the following primary antibodies: anti-CD133 antibody (1:100; Proteintech), anti-CD44 (1:100, Proteintech), anti-c-MYC (1:100; Proteintech), and anti-Ki-67 (MXB, Fuzhou, China).
Immunofluorescence and confocal microscopy were performed as previously reported [19 (link), 20 (link)]. Images were captured by using a Zeiss confocal microscope.
Immunofluorescence and confocal microscopy were performed as previously reported [19 (link), 20 (link)]. Images were captured by using a Zeiss confocal microscope.
4
Quantitative Protein Analysis by Western Blot
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Protein extraction and Western blotting were performed according to standard procedures. Primary antibodies were used as follows: anti-c-MYC (1:1,000, Proteintech), anti-EZH2, anti-SMAD2, anti-RB, anti-CDK4, anti-CDK6, anti-CCND1, anti-CCND2 and anti-CCND3 (1:1,000, Cell Signaling Technology) and anti-Actin (1:3,000, Sigma). The membrane was then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:3,000 Cell Signaling Technology). The intensity of protein bands was quantified by using the Image-J software package.
5
Western Blot Analysis of BCa Cells
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A mixture of RIPA lysis buffer (Beyotime, China) and protease inhibitor (CMBIO, China) at a ratio of 100:1 was used to lyse BCa cells. Samples containing 30 μg of total protein were separated by 10% SDS–PAGE, and then the protein was transferred onto PVDF membranes for 2 hours. After blocking with 5% nonfat milk in TBST for 1 h at room temperature, anti-BARX2 (Bioss, China; cat. No. bs-19273R-3; dilution: 1:800), anti-c-MYC (Proteintech, China; cat. NO. 10828-1-AP; dilution: 1:5000), anti-β-catenin (Proteintech, China; cat. NO. 51067-2-AP; dilution: 1:5000), anti-GAPDH (Proteintech, China; cat. NO. 10494-1-AP; dilution: 1:10000), anti-Histone H3.1 (Beijing Ray Antibody Biotech, China; cat. NO. RM2005L; dilution 1:5000) and anti-β-actin (Proteintech, China; cat. No. 66009-1-Ig; dilution: 1:10000) antibodies were used to incubate PVDF membranes at 4° C overnight. The next day, the membranes were incubated with HRP-coagulated anti-rabbit or anti-mouse antibody at room temperature for 1 h.
6
Western Blot Analysis of IGF2BP1, c-Myc, and β-Actin
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The cells were lysed using RIPA lysates (Solarbio, Beijing, China) with protease inhibitor (Beyotime, Beijing, China). Total proteins in the lysates were separated by 10% SDS–polyacrylamide gel electrophoresis and dotted to a polyvinylidene difluoride membrane (Millipore, MA, USA). We incubated the membranes with 5% skim milk powder for 1 h to block the non-specific binding at room temperature (21–25℃). Then the membrane was incubated overnight with the primary antibody. anti-IGF2BP1, anti-c-Myc, and anti-β-actin were purchased from Proteintech (Wuhan, China).
7
Immunohistochemical Evaluation of Stem Cell Markers
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The procedure of IHC and ICC staining was shown in the previous studies [52 (link)]. LGR6 staining was evaluated blindly and independently by two pathologists. The score for staining intensity (0 = no staining, 1 = light brown, 2 = brown, and 3 = dark brown) and staining frequency (0 = 0–10%, 1 = 11–25%, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100%) were multiplied to obtain the immunoreactivity score (IRS). The IRS 0–3 was deemed negative, 4–6 weak positive, and >6 strong positive. Two different pathologists evaluated all the specimens in a blinded manner. The antibodies used were as follows: anti-LGR6 (#MAB8458, R&D Systems, USA), anti-β-catenin (#sc-7963, Santa Cruz, USA), anti-TCF7L2 (#sc-166699, Santa Cruz, USA), anti-c-Myc (#10828-1-AP, Proteintech, China), OCT4 (#sc-5279, Santa Cruz, USA), anti-SOX2 (#3579, Cell Signaling Technology, USA), anti-KLF4 (#sc-20691, Santa Cruz, USA), anti-ALDH1A1 (#sc-374149, Santa Cruz, USA), anti-LGR5 (#PAB2591, Abnova, Taiwan), anti-LGR4 (#sc-390630, Santa Cruz, USA).
8
Antibody-Mediated Apoptosis Regulation
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Antibodies and reagents used in the study are listed as follows: anti- Cleaved Caspase3 was purchased from Cell Signaling Technology (1:1000) (Boston, America) Anti-Histone H3 (acetyl K27) (ab4729, 1:2000), anti-Cyclin D2 (ab230883, 1:2000) were purchased from Abcam company (Cambridge, England). Anti-c-Myc (10828-1-AP, 1:1000), anti-CDK4 (66950-1-Ig, 1:1000), anti-BOP1 (28366-1-AP, 1:1000), anti-NOP56 (18181-1-AP, 1:1000), anti-GAPDH (60004-1-Ig, 1:1000), anti-P21 (60214-1-Ig, 1:1000), anti-HDAC7 (26207-1-AP, 1:1000) and anti- Histone H3 (17168-1-AP, 1:1000) were purchased from Protein-tech Group (Wuhan, China). Dissolved Z31216525, Z46582199, Z165155756 and Z234820564 (Enamine Ltd, Ukraine) in DMSO separately at a concentration of 10 mM.
9
Comprehensive Protein Analysis Workflow
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Western blotting was performed as previously described,35 (link) using anti-CDK5, anti-FAK, anti-p-FAK (Ser732), anti-PAK1, anti-p-PAK1 (Thr212), anti-ERK5, anti-p-ERK5 (Thr218/Tyr220) (Abcam), anti-p-ERK5 (Thr732), anti-p35 (Cell Signaling Technology, Danvers, MA, USA; anti-p-ERK5 Thr732 was custom-made from CST), anti-c-fos, anti-c-jun (Bioworld Technology, Louis Park, MN, USA), anti-c-myc, anti-VEGFA and anti-MMP1 antibodies (Proteintech, Chicago, IL, USA). Loading control was used with a mouse anti-β-Actin monoclonal antibody (Proteintech).
10
Analyzing Cisplatin-Induced Apoptosis Pathways
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Cells transfected with either control siRNA or siR-β-catenin were cultured with or without cisplatin for 24 h. Subsequently, the cells were lysed with radioimmunoprecipitation assay lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) with protease and phosphatase inhibitors. Protein content was determined by a Bradford protein kit (cat. no. P0012S; Beyotime Institute of Biotechnology). The proteins (30 µg/lane) were separated by 10% SDS-PAGE (cat. no. P0012A; Beyotime Institute of Biotechnology) and transferred onto PVDF membranes (EMD Millipore). Following blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following antibodies: Anti-β-catenin (cat. no. 17565-1-AP; 1:4,000; ProteinTech Group, Inc.), anti-c-Myc (cat. no. 10828-1-AP; 1:2,000; ProteinTech Group, Inc.), anti-cyclin D1 (cat. no. 26755-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-caspase 3 (cat. no. 19677-1-AP; 1:600; ProteinTech Group, Inc.), anti-caspase 9 (cat. no. 10380-1-AP; 1:800; ProteinTech Group, Inc.) and anti-β-actin (cat. no. 20536-1-AP; 1:800; ProteinTech Group, Inc.), followed by horseradish peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (H+L) (cat. no. SA00001-9; 1:4,000; ProteinTech Group, Inc.) at 4°C for 2 h. The band intensity was tested using ImageJ v.1.47 software (National Institutes of Health).
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