SDS-PAGE was performed using 4–12% Bis-Tris gels (Novex). After running the gels, proteins were transferred onto nitrocellulose membrane (VitaScientific, DBOC80014). Western blotting was performed according to a standard protocol. SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, 34078), or SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, 34580), or One Component Chemiluminescent Substrate (Rockland, UniGlow-0100) was used for the detection. Protein levels were quantified using Gene Tools software (version 3.8.8.0) from SynGene (Frederick, MD) or Image Lab software (version 6.0.0) from Bio-Rad Laboratories, Inc (Hercules, CA). Antibodies were as follows: anti-HA (12CA5) mouse (Roche, 11583816001), anti-HA rabbit (Sigma, H6908), anti-c-Myc (A-14) (Santa Cruz Biotechnology, sc-789), anti-Smt3 (y-84) (Santa Cruz Biotechnology, sc-28649), and anti-Tub2 antibodies (Basrai laboratory). Anti-HA was used at a dilution from 1:1000 to 1:5000, and anti-c-Myc, anti-Smt3, and anti-Tub2 were used at a dilution from 1:3000 to 1:5000. Secondary antibodies were ECL Mouse IgG, HRP-linked whole Ab (GE Healthcare Life Sciences, NA931V) or ECL Rabbit IgG, HRP-linked whole Ab (GE Healthcare Life Sciences, NA934V) at 1:5000 dilution.
Anti c myc a 14
Manufactured by Santa Cruz Biotechnology
Anti-c-Myc (A-14) is a primary antibody that recognizes the c-Myc protein. It is produced in rabbit and can be used for various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti smt3 y 84, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Genetools software, by Syngene (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Anti c myc 9e10, by Merck Group (1 mentions)
Protran nitrocellulose membrane, by Cytiva (1 mentions)
Mops buffer, by Thermo Fisher Scientific (1 mentions)
Ibind flex western device, by Thermo Fisher Scientific (1 mentions)
Nupage lds buffer, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
O phenylenediamine dihydrochloride reagent, by Merck Group (1 mentions)
3 protocols using anti c myc a 14
1
Quantitative Western Blot Analysis
2
Western Blot Analysis of Protein Extracts
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Western blot analysis was performed with native protein extracts, prepared as described above, diluted to 6 µg µl−1 and mixed with equal volume of 2× protein sample buffer (2× NuPAGE LDS buffer (Life Technologies), 100 mM DTT, 4% (w/v) SDS). Samples were then incubated for 10 min at 75 °C and 10 μl (30 μg) of samples was loaded onto a 4–12% NuPAGE Bis-Tris gel (Life Technologies, NP0321BOX). Electrophoresis was done in 1× MOPS buffer (Life technologies, NP0001) at 160 V for 60 min. Proteins were transferred to Protran nitrocellulose membranes (Whatman) in western transfer buffer (3.03 g l−1 Tris base, 14.4 g l−1 glycine, 20% (v/v) methanol) at 100 V for 1 h. Blots were blocked and washed with iBind Flex Western Device (Life Technologies, SLF20002). Lsm and Sm blots were probed with mouse monoclonal anti-c-Myc 9E10 (Sigma-Aldrich, M4439) at 1:5000 dilution and horse-radish peroxidase-conjugated goat anti-mouse IgG (H+L) at 1:5000 (Thermo Scientific, 31430). Trt1 blot was probed with rabbit polyclonal anti-cMyc A14 (Santa Cruz Biotechnologies, sc-789) at 1:400 dilution and horse-radish peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1:4000 (Thermo Scientific, 31460). Blots were reprobed with mouse anti-α-tubulin (Sigma-Aldrich, T5168).
3
GLUT4 Trafficking Assay in L6 Myoblasts
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For details, see Wang et al [26 (link)]. Briefly, L6-GLUT4myc myoblasts or myotubes were serum-starved (4 h), treated with M-3 (250 nmol/l), stimulated with insulin (20 or 100 nmol/l) or vehicle at 37°C for 30 min, and fixed in 3% paraformaldehyde. Anti-c-myc (A14; Santa Cruz, Heidelberg, Germany; 1:100) primary antibody was applied (1 h), and peroxidase-conjugated rabbit anti-mouse secondary antibody (1:1,000) in 3% goat serum was added, followed by 1 ml o-phenylenediamine dihydrochloride (OPD reagent; Sigma), and incubated (in the dark) for 30 min. The reaction was stopped with 250 μl 3 mol/l HCl, and absorbance of the supernatant fraction was measured.
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