Equivalent amounts of protein were suspended in protein sample buffer (50 mM Tris pH6.8, 5% SDS, 10% glycerol, 5% β-mercaptoethanol, 25mM NaF, 1mM Na3VO4, 5mM β-glycerophosphate, 1mM PMSF, 50 μM Leupeptin, 100 μM Pepstatin A), resolved by electrophoresis in Bis-Tris polyacrylamide gels and transferred to polyvinylidene fluoride membranes by electroblotting. Membranes were blocked in TNET (10 mM Tris pH 7.5, 50 mM NaCl, 2.5 mM EDTA pH 8.0, 0.1% tween) supplemented with 5% nonfat dry milk. Primary antibodies used in these experiments were against: HA (#12013819001, Roche), FLAG (F3165, Sigma), SMCX (NB100-55328, Novus Biologicals), EP400 (A300-541A, Bethyl Laboratories), Brd4 (Schweiger et al., 2006 (link)), TIP60 (sc-16623, Santa Cruz Biotechnology) and actin (MAB1501, Millipore). Bound antibodies were detected with horseradish peroxidase conjugated anti-rabbit or anti-mouse secondary antibodies. For detection of actin and GAPDH, membranes were incubated with an AlexaFluor 680 conjugated goat anti-mouse IgG secondary antibody (Molecular Probes/Invitrogen) and labeled proteins visualized using a LI-COR Odyssey Infrared Imaging System.
Ep400
Manufactured by Fortis Life Sciences
The EP400 is a laboratory instrument designed for electrophoresis. It is used to separate and analyze macromolecules, such as proteins or nucleic acids, based on their size and electrical charge. The EP400 provides a controlled environment and precise voltage regulation for reliable and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Actin, by Thermo Fisher Scientific (1 mentions)
Actin, by Merck Group (1 mentions)
Alexafluor 680 conjugated goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Tip60, by Santa Cruz Biotechnology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
G box chemi system, by Syngene (1 mentions)
Ruvbl2, by Fortis Life Sciences (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
H3k4me3, by Merck Group (1 mentions)
Anti ha magnetic beads, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
2 protocols using ep400
1
Protein Characterization by Western Blotting
2
Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used: Ab5 and Ab3 [23 (link), 73 (link)]; HA (Abcam); EP400, RUVBL2 (Bethyl); MAX, KAT5, DMAP1, MNT (Santa Cruz); MYCL (R&D Systems); ING3 (Sigma); PPP2CA (BD Biosciences); and H3K4me3 (Millipore; 07–473).
Cell lysates were prepared in EBC Lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA, 1 mM β-Mercaptoethanol and freshly added protease inhibitor and phosphatase inhibitor cocktail). Immunoprecipitations were performed with protein G Dynabeads (Life Technologies) mixed with immunoprecipitation antibodies or anti-HA magnetic beads (Pierce Biotechnology). After overnight incubation on a rotating apparatus at 4°C, magnetic beads were washed with high salt wash buffer (50 mM Tris pH 7.4, 300 mM NaCl, 0.5% NP-40, 0.5 mM EDTA) five times. Bound proteins were eluted from magnetic beads with 2x Laemmli sample buffer (Bio-Rad). After electrophoresis, the separated proteins were transferred to PVDF membrane and blotted. Immunoblots were developed using Clarity Western ECL substrate (Bio-Rad) and imaged with G:BOX Chemi system (Syngene).
Cell lysates were prepared in EBC Lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA, 1 mM β-Mercaptoethanol and freshly added protease inhibitor and phosphatase inhibitor cocktail). Immunoprecipitations were performed with protein G Dynabeads (Life Technologies) mixed with immunoprecipitation antibodies or anti-HA magnetic beads (Pierce Biotechnology). After overnight incubation on a rotating apparatus at 4°C, magnetic beads were washed with high salt wash buffer (50 mM Tris pH 7.4, 300 mM NaCl, 0.5% NP-40, 0.5 mM EDTA) five times. Bound proteins were eluted from magnetic beads with 2x Laemmli sample buffer (Bio-Rad). After electrophoresis, the separated proteins were transferred to PVDF membrane and blotted. Immunoblots were developed using Clarity Western ECL substrate (Bio-Rad) and imaged with G:BOX Chemi system (Syngene).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!