Total protein was extracted from infected cells or tumors, respectively. The protein concentration was measured by BCA Protein Assay Kit (Solarbio, Shanghai, China). 20 μL protein samples were resolved by 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Protein bands were incubated with primary antibodies at 4°C overnight after being blocked with 5% skim milk. Then, membranes were incubated with HRP labeled secondary antibodies (Solarbio; 1:3000) at 37°C for an hour. Protein probes were imaged with an ECL reagent (Solarbio). The primary antibodies were ACSM3 (Proteintech. Wuhan, China; 1:500), Integrin β1 (Proteintech; 1:500), E-cadherin (ABclonal, Shanghai, China; 1:1000), cyclin D1 (ABclonal; 1:500), p-AKT (ABclonal; 1:500), AKT (ABclonal; 1:1000), Vimentin (ABclonal; 1:500), c-Myc (ABclonal; 1:500), and GAPDH (Proteintech; 1:10000).
C myc
Manufactured by ABclonal
Sourced in China
The C-Myc antibody is a laboratory reagent used to detect the presence and expression of the c-Myc protein, which is a transcription factor involved in cell growth and proliferation. The antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to study the expression and localization of the c-Myc protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Gapdh, by Proteintech (2 mentions)
Gapdh, by ABclonal (2 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
P akt, by ABclonal (1 mentions)
Ecl reagent, by Solarbio (1 mentions)
Acsm3, by Proteintech (1 mentions)
Integrin β1, by Proteintech (1 mentions)
Cyclin d1, by ABclonal (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Vimentin, by ABclonal (1 mentions)
E cadherin, by ABclonal (1 mentions)
Hrp conjugated secondary antibody, by Solarbio (1 mentions)
Ecl plus kit, by Solarbio (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Bca protein concentration assay kit, by Solarbio (1 mentions)
Skim milk, by Sangon (1 mentions)
β catenin, by ABclonal (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
12 protocols using c myc
1
Western Blot Analysis of Proteins
2
Western Blot Analysis of Protein Expression
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Total proteins were extracted using RIPA buffer (Solarbio, Beijing, China) containing 1% PMSF (Solarbio, Beijing, China). Protein concentrations were determined using the BCA protein concentration assay kit (Solarbio, Beijing, China). Protein samples (10–20 µg) were diluted to the same volume with Loading Buffer and then separated by 5% SDS-PAGE before transferring to PVDF membranes (Millipore, Billerica, MA, US). The membranes were blocked in 5% skim milk (Sangon, Shanghai, China) for 1 h and then incubated with the specific primary antibodies overnight at 4 °C. The primary antibodies are as follows: KHDRBS3 (Santa Cruz Biotechnology, sc-374,461, Shanghai, China; 1:500); c-Myc (ABclonal, A1309, Shanghai, China; 1:500); GLUT1 (Affbiotech, AF5462, Changzhou, China; 1:500); LDHA (ABclonal, A1146, Shanghai, China; 1:1000); 14-3-3ζ (Affbiotech, AF6356, Changzhou, China; 1:1000); GAPDH (Proteintech, 60004-1-Ig, Wuhan China; 1:10000). After incubation with HRP-conjugated secondary antibodies (Solarbio, Beijing, China), the bands were visualized using ECL Plus kit (Solarbio, Beijing, China).
3
Chondrosarcoma Cell Culture Protocol
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SW1353 chondrosarcoma cell line (Cell Bank of Chinese Academy of Sciences; Shanghai, China) was cultured with Dulbeccoʼs modified Eagleʼs medium (DMEM) containing 1% double antibody and 10% fetal bovine serum (FBS) (Gibco; USA) in a cell incubator with 5% CO2 at 37°C. The complete DMEM was replaced every other day. SA (Sigma-Aldrich; Shanghai, China) was dissolved with dimethyl sulfoxide (Solarbio; Beijing, China). Rabbit polyclonal antibodies against Bax, Bcl-2, WNT3A, β-Catenin, c-Myc, and GAPDH were obtained from ABclonal Technology (Shanghai, China). Rabbit monoclonal antibodies against N-cadherin and E-cadherin were acquired from cell signaling technology (USA). Other reagents were purchased from Sigma-Aldrich (Shanghai, China) unless additional described.
4
Antibody Panel for Protein Analysis
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The antibodies used in this study are as follows. KIF2C: sc-81305, Santa Cruz; TBC1D7: #14949, Cell Signaling Technology; TSC1: #6935, Cell Signaling Technology; TSC2: #4308, Cell Signaling Technology; mTOR: #2983, Cell Signaling Technology; p-mTOR: #5536, Cell Signaling Technology; p70 S6K: #2780, Cell Signaling Technology; p-p70 S6K: #9234, Cell Signaling Technology; 4EBP1: #9644, Cell Signaling Technology; p-4EBP1: #2855, Cell Signaling Technology; β-catenin: #8480, Cell Signaling Technology; cyclin D1: #2922, Cell Signaling Technology; c-Myc: A1309, Abclonal; GAPDH: 60004-1-Ig, Proteintech.
5
Culturing Lung Cancer Cell Lines
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6
Protein Expression Analysis Protocol
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Primary antibodies used were RB (Cell Signaling Technology, # 9309, 1:1000), RB (Cell Signaling Technology, # 9313, 1:1000), E2F1 (Proteintech, #66515-1-Ig, 1:1000), Phospho-Rb (Ser795) (Cell Signaling Technology, # 9301S, 1:1000), RBL1 (Proteintech, #13354-1-AP, 1:1000), RBL2 (Proteintech, #27251-1-AP, 1:1000), Cyclin B1 (ABclonal, # A19037, 1: 1000), RNF40 (ABclonal, # A6443, 1: 1000), LRPPRC (ABclonal, # A3365, 1: 1000), CDK4 (ABclonal, # A11136, 1: 1000), CDK6 (ABclonal, # A0106, 1: 1000), KATNA1 (ABclonal, # A16491, 1: 1000), c-MYC (ABclonal, # A1309, 1: 1000), KLHL42 (Invitrogen, # PAS-54292, 1: 500), Vinculin (Santa Cruz, # sc-73614, 1:1000), β-Actin (Cell Signaling Technology, # 4970, 1:1000), Myc (Santa Cruz, # sc-40, 1:1000), HA (Cell Signaling Technology, # 3724, 1:1000), Flag (MBL, #M185-7, 1:1000), Rabbit IgG (ABclonal, # AS014, 1:3000), Mouse IgG (ABclonal, # AS003, 1:3000).
7
Western Blot Analysis of Protein Expression
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Cells were harvested, washed, and lysed for total protein extraction. Equal quantities of protein extract were injected into sodium dodecyl sulfate–polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with alkaline phosphatase-conjugated secondary IgG antibody. The bands were incubated with a DAB kit and analyzed with an imaging system. Antibodies against β-actin (#AC026) and c-Myc (#A19032) were purchased from ABclonal (Wuhan, China).
8
Western Blot Analysis of Protein Expression
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Total protein was extracted using RIPA lysis (Boster, China) buffer containing protease and phosphatase inhibitors. The BCA Protein Assay Kit (Boster) was used to determine protein concentrations. Equal quantities of proteins were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and transferred to polypropylene difluoride membranes (Millipore, USA). At room temperature, the membranes were blocked in 5% nonfat milk for 1 hour. Primary antibodies against GADPH (#10494-1-AP, 1:4 000, Proteintech, China), DHCR24 (#2033S, 1:1 000, Cell Signaling Technology, USA), SIRT1 (#8469S, 1:1 000, Cell Signaling Technology), P16 (#ab51243, 1:1 000, Abcam, UK), caveolin-1 (#A1555, 1:1 000, ABclonal, China), p-ERK (#4370T 1:1 000, Cell Signaling Technology), ERK (#4695T 1:1 000, Cell Signaling Technology), p-c-myc (#AP0989, 1:1 000, ABclonal), c-myc (#A19032, 1:1 000, ABclonal), and eNOS (#32027S, 1:1 000, Cell Signaling Technology) were incubated at 4°C overnight. The membranes were then incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5 000; Promotor, China). Finally, chemiluminescence was detected with an enhanced chemiluminescence kit (Beyotime).
9
Comprehensive Analysis of Protein Expression
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Cells were lysed using RIPA lysis buffer containing protease inhibitors to obtain protein. A BCA Protein Assay Kit was used to determine the protein concentrations. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Subsequently, the membranes were blocked with 5% BSA blocking reagent for 1 hour at RT and incubated with primary antibodies overnight at 4° C. The next day, the membranes were washed and incubated for 1 hour at RT with secondary antibodies. Finally, the proteins were analysed using the ECL Plus kit. The antibodies used included the following: CUEDC1 (ab58696, Abcam, 1:1000); Flag antibody (#14793, Cell Signaling Technology, 1:50); E-cadherin (ab40772, Abcam, 1:1000); N-cadherin (ab18203, Abcam, 1:1000); Vimentin (ab92547, Abcam, 1:1000); Smurf2 (ab53316, Abcam, 1:1000); TβRI (A0708, ABclonal, 1:1000); Snail (#3879, Cell Signaling Technology, 1:1000); p-Smad2 (Ser465/467, #3108, Cell Signaling Technology, 1:1000); p-Smad3 (Ser423/425, #9520, Cell Signaling Technology, 1:1000); CyclinD1 (26939-1-AP, Proteintech, 1:1000); CDK4 (#12790, Cell Signaling Technology, 1:1000); C-myc (A1309, ABclonal, 1:1000); Bax (A15646, ABclonal, 1:1000); Bcl-2 (26593-1-AP, Proteintech, 1:1000); and β-actin (TA-09, ZSGB-BIO, 1:1500).
10
Western Blot Analysis of Signaling Pathways
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The cells were incubated with various concentrations of AS-605240 or/and Melphalan for 48 h. Whole cell lysate was lysed in RIPA buffer (Beyotime Biotechnology, Beijing, China). The total protein concentration was quantified using a BCA Protein Assay Kit (Beyotime Biotechnology, Beijing, China). Blots were developed using the Efficient Chemiluminescence Kit (GENVIEW) and SageCapture imaging System (SAGECREATION). Primary antibodies were c-Myc (1:1000; ABclonal, No.A1309), Bax (1:1000; Proteintech, No. 50599-2-Ig), Bcl-2 (1:1000; Proteintech, No. 12789-1-AP), PIK3CG (1:1000; CST, No.5405), AKT (1:1000; CST, No.4691), p-AKT (1:1000; CST, No.4060), RELA (1:1000; ABclonal, No.A16271), p-RELA(1:1000; Wanlei, No.102132169), and Vimentin (1:1000; CST, No.5741S). Blots were reprobed with anti-β-actin antibody (1:2000; ABclonal, No.AC004) as a loading control. The secondary antibodies used for Western blot were Goat Anti-Mouse IgG (1:4000; AB clonal, No. AS003) and Goat Anti-Rabbit IgG (1:4000; Abbkine, No.A21020).
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