After being transfected for 48 h, cells were collected by centrifugation, followed by total protein extraction. The proteins were quantified by BCA (bicinchoninic acid) protein assay kit (Jianglaibio, Shanghai, China) and separated by 12% SDS-PAGE. Then, proteins were transferred to the PVDF membrane and blocked with 5% skim milk. The membranes were incubated with primary antibodies of MMP-2 monoclonal antibody (1 : 500, Invitrogen), MMP-9 polyclonal antibody (1 : 1000, Invitrogen), Cyclin D1 monoclonal antibody (1 : 200, Invitrogen), c-myc monoclonal antibody (1 : 200, Invitrogen), JAK1 monoclonal antibody (1 : 1000, Invitrogen), p-JAK1 polyclonal antibody (1 : 1000, Invitrogen), STAT3 monoclonal antibody (1 : 5000, Invitrogen), p-STAT3 polyclonal antibody (1 : 1000, Invitrogen), and GAPDH monoclonal antibody (1 : 1000, Invitrogen) at room temperature overnight, followed by incubation with the secondary antibody at 37°C for 90 min. Finally, proteins were detected with the ECL method under a multi-imager (Bio-Rap, Hercules, CA, USA).
C myc monoclonal antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The C-myc monoclonal antibody is a laboratory reagent used for the detection and analysis of the c-myc protein. It is a highly specific antibody that binds to the c-myc protein, which is a transcription factor involved in cellular processes such as cell growth and proliferation. The C-myc monoclonal antibody can be used in various laboratory techniques, including Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of the c-myc protein in different cell types and tissues.
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Lab products found in correlation
Gapdh monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Gfp sc 9996, by Santa Cruz Biotechnology (1 mentions)
Affinipure donkey anti rabbit or donkey anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Ha tag, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Molecular imager chemidoc xrs, by Bio-Rad (1 mentions)
Monoclonal anti gfp, by Roche (1 mentions)
Goat anti mouse igg h l hrp conjugate, by Bio-Rad (1 mentions)
Anti ha, by Roche (1 mentions)
Goat anti rabbit igg h l hrp conjugate, by Bio-Rad (1 mentions)
3 protocols using c myc monoclonal antibody
1
Protein Expression Analysis by Western Blot
2
Protein Extraction and Western Blot Analysis
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Cells were harvested and resuspended in lysis buffer (1% Triton X-100, 100 mM NaCl, 50 mM Tris, pH 7.8) to incubate for 1 h on ice. Cell lysates were clarified by centrifugation, and protein concentration was determined by BCA protein assay kit. After mixing with 2× Laemmli buffer, samples were subjected to 7.5% or 10% SDS-PAGE. Proteins were then transferred to a nitrocellulose membrane (0.45 µm, #1620115; Bio-Rad) followed by blocking with 5% (wt/vol) skim milk in TBS plus Tween 20 for 1 h at RT. Incubation with primary antibody (1:1,000) was performed overnight at 4°C after three washes for 5 min each in TBS plus Tween 20. Primary antibodies included in this study were mouse monoclonal c-myc monoclonal antibody (clone 9E10, 13-2500; Invitrogen), GFP (sc-9996; Santa Cruz Biotechnology), rabbit polyclonal to VMP1 (12929S; Cell Signaling Technology), GAPDH (2118; Cell Signaling Technology), β-actin (4970; Cell Signaling Technology), and HA-tag (3724S; Cell Signaling Technology). Secondary antibodies were peroxidase-conjugated AffiniPure donkey anti-rabbit or donkey anti-mouse IgG (H+L; Jackson ImmunoResearch Laboratories) used at a 1:5,000 dilution at RT for 1 h. The bound antibodies were detected by ECL Western Blotting Detection Reagent (GE Healthcare or Merck Millipore) and visualized with Molecular Imager® ChemiDoc™ XRS+ (Bio-Rad).
3
Protein Extraction and Immunoblotting in Yeast
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The protocol was adapted from (Choudhary and Schneiter, 2012 (link)). Briefly, proteins were extracted from three OD600 units of yeast cells by NaOH followed by precipitation with 10% TCA. To analyze proteins in the culture supernatant, proteins from 20 ml media of an overnight grown culture were precipitated by 10% TCA.
The primary antibodies used were: anti-HA (rat, 1:2000, Roche #11867423001), c-Myc monoclonal antibody (mouse, 1:5000, Invitrogen #13-2500), monoclonal anti-GFP (mouse, 1:2000, Roche #11814460001), anti-Kar2 (rabbit, 1:5000, Randy Schekman, University of California at Berkeley, CA, USA). As secondary antibodies goat anti-rat IgG antibody, horseradish peroxidase (HRP) conjugate (1:10,000, Merck #AP136P), goat anti-rabbit IgG (H+L)-HRP conjugate (1:10,000, Bio-Rad #1706515) and goat anti-mouse IgG (H+L)-HRP conjugates (1:10,000, Bio-Rad #1706516) were employed.
The primary antibodies used were: anti-HA (rat, 1:2000, Roche #11867423001), c-Myc monoclonal antibody (mouse, 1:5000, Invitrogen #13-2500), monoclonal anti-GFP (mouse, 1:2000, Roche #11814460001), anti-Kar2 (rabbit, 1:5000, Randy Schekman, University of California at Berkeley, CA, USA). As secondary antibodies goat anti-rat IgG antibody, horseradish peroxidase (HRP) conjugate (1:10,000, Merck #AP136P), goat anti-rabbit IgG (H+L)-HRP conjugate (1:10,000, Bio-Rad #1706515) and goat anti-mouse IgG (H+L)-HRP conjugates (1:10,000, Bio-Rad #1706516) were employed.
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