Genomic DNA and methylated DNA standards (Cat. #55008; Active Motif) were heat- denatured in 0.4 M NaOH (Sigma) & 10 mM EDTA (Sigma) at 100 °C for 10 min and chilled on ice for 5 min. Samples were two-fold serially diluted and applied to a positively charged nylon membrane (Biodyne B; Pall Life Sciences) using the Bio-dot microfiltration apparatus (Biorad). Membranes were UV-crosslinked at 12,0000 μJ/cm2 (CL1000; UVP), and blocked in TBS, 0.1% Tween-20 (TBST), 5% milk for 1 hr. Primary antibodies against 5-mC (1:1000; cat. 39649; Active Motif) were incubated with the membrane in TBST overnight. Dot blots were washed 3X in TBST, incubated in HRP-conjugated IgG secondary antibodies (GE healthcare) for 1hr, and detected by standard ECL methods (Supersignal West Pico; Thermo Fisher Scientific).
Hrp conjugated igg secondary antibodies
Manufactured by GE Healthcare
HRP-conjugated IgG secondary antibodies are proteins designed to bind and detect target primary antibodies. They are labeled with Horseradish Peroxidase (HRP), an enzyme that can catalyze a colorimetric reaction for visualization purposes. These antibodies provide a versatile tool for immunoassays and immunohistochemistry applications.
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Lab products found in correlation
2 protocols using hrp conjugated igg secondary antibodies
1
Quantitative DNA Methylation Analysis
2
Quantitative DNA Methylation Analysis
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Genomic DNA and methylated DNA standards (Cat. #55008; Active Motif) were heat- denatured in 0.4 M NaOH (Sigma) & 10 mM EDTA (Sigma) at 100 °C for 10 min and chilled on ice for 5 min. Samples were two-fold serially diluted and applied to a positively charged nylon membrane (Biodyne B; Pall Life Sciences) using the Bio-dot microfiltration apparatus (Biorad). Membranes were UV-crosslinked at 12,0000 μJ/cm2 (CL1000; UVP), and blocked in TBS, 0.1% Tween-20 (TBST), 5% milk for 1 hr. Primary antibodies against 5-mC (1:1000; cat. 39649; Active Motif) were incubated with the membrane in TBST overnight. Dot blots were washed 3X in TBST, incubated in HRP-conjugated IgG secondary antibodies (GE healthcare) for 1hr, and detected by standard ECL methods (Supersignal West Pico; Thermo Fisher Scientific).
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