Western blot analysis was performed with whole cell lysate. The cell monolayer was rinsed three times with ice-cold PBS prior to collecting cells with a cell lifter (VWR) and pelleting by centrifugation. The cell pellets were lysed in 20 mM HEPES pH8.0, 100 mM NaCl, 1 mM EDTA and 1% Tween20 containing protease inhibitor cocktail (ROCHE) on ice for 30 minutes, sonicated, and cell debris was cleared by centrifugation. The protein concentration of the supernatant was quantified using the micro BCA protein assay kit (Pierce). About 70–100 μg of total protein was separated on 4–12% SDS-PAGE gel (Invitrogen) and transferred to PVDF membranes (GE Healthcare) followed by incubation with anti-rabbit-DNMT3A antibody (Abcam ab188470 lot:GR224165–13) and anti-rabbit-GAPDH antibody (Cell Signaling 2118 lot: 10) as a loading control. All blots were developed using the ECL reaction kit (Bio-Rad) according to the manufacturer’s instructions.
Ecl reaction kit
Manufactured by Bio-Rad
The ECL reaction kit is a laboratory product designed for use in Western blot analysis. It provides the necessary reagents for performing enhanced chemiluminescent (ECL) detection of proteins that have been transferred to a membrane. The kit includes solutions for protein detection, but does not include any interpretation or recommendations for its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Cell lifter, by Avantor (2 mentions)
Anti rabbit dnmt3a antibody, by Abcam (2 mentions)
Rabbit anti gapdh antibody, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Sds page gel, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
2 protocols using ecl reaction kit
1
Western Blot Analysis of DNMT3A Protein
2
Western Blot Analysis of DNMT3A Protein
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Western blot analysis was performed with whole cell lysate. The cell monolayer was rinsed three times with ice-cold PBS prior to collecting cells with a cell lifter (VWR) and pelleting by centrifugation. The cell pellets were lysed in 20 mM HEPES pH8.0, 100 mM NaCl, 1 mM EDTA and 1% Tween20 containing protease inhibitor cocktail (ROCHE) on ice for 30 minutes, sonicated, and cell debris was cleared by centrifugation. The protein concentration of the supernatant was quantified using the micro BCA protein assay kit (Pierce). About 70–100 μg of total protein was separated on 4–12% SDS-PAGE gel (Invitrogen) and transferred to PVDF membranes (GE Healthcare) followed by incubation with anti-rabbit-DNMT3A antibody (Abcam ab188470 lot:GR224165–13) and anti-rabbit-GAPDH antibody (Cell Signaling 2118 lot: 10) as a loading control. All blots were developed using the ECL reaction kit (Bio-Rad) according to the manufacturer’s instructions.
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