HEK293T cells were transiently cotransfected with the indicated plasmids. After transfection for 48 h, the cells were lysed in whole-cell lysis buffer (50 mM Tris-HCl pH=7.6, 150 mM NaCl, and 1.0% NP-40) containing a protease inhibitor cocktail (Sigma-Aldrich). Next, 500 µg of each cell lysates was incubated with protein A/G PLUS-agarose beads (Abmart, Shanghai, China) at 4 °C for 2 h. Then, the lysates were incubated with 30 µl of anti-Flag M2 beads (Sigma-Aldrich) at 4 °C overnight with constant shaking. The samples were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, USA), and analyzed by Western blot using an Flag-HRP antibody (1:10000; RRID:AB_439702) and an anti-Myc-HRP antibody (1:5000; Thermo Scientific, RRID:AB_779857).
Anti myc hrp antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-Myc-HRP antibody is a conjugate of an anti-Myc antibody and horseradish peroxidase (HRP). It is used to detect and quantify proteins that have been tagged with the Myc epitope.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protein a g plus agarose beads, by Abmart (1 mentions)
Anti flag m2 bead, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase, by Cell Signaling Technology (1 mentions)
Anti lamin a c antibody, by Cell Signaling Technology (1 mentions)
Anti v5 hrp antibody, by Thermo Fisher Scientific (1 mentions)
Anti jab1 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2 horse radish peroxidase antibody, by Merck Group (1 mentions)
2 protocols using anti myc hrp antibody
1
Co-Immunoprecipitation of Flag- and Myc-tagged Proteins
2
Western Blot Analysis of Protein Markers
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Western blot analyses were performed as described previously 15 . Primary antibodies used for western blot and dilution are as follows: anti‐FLAG M2–horse radish peroxidase (HRP) antibody (Sigma‐Aldrich, 1 : 1000), anti‐myc–HRP antibody (Thermo Fisher Scientific, 1 : 5000) and anti‐V5–HRP antibody (Thermo Fisher Scientific, 1 : 5000), anti‐Lamin A/C antibody (Cell Signaling Technology, Danvers, MA, USA, 1 : 1000), anti‐glyceraldehyde‐3‐phosphate dehydrogenase (Cell Signaling Technology, 1 : 500), anti‐p27kip1 antibody (MBL, 1 : 1000), anti‐JAB1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA, 1 : 200), anti‐Cdk2 antibody (Santa Cruz Biotechnology, 1 : 200), and anti‐cyclin E antibody (MBL, 1 : 1000). Anti‐mouse IgG HRP‐linked and anti‐rabbit IgG HRP‐linked (Cell Signaling Technology, 1 : 5000) secondary antibodies were used. Data were analyzed using lumivision analyzer 400 software (Aisin Seiki, Kariya, Aichi, Japan).
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