Stat5b Antibody (C-8): sc-377069

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Investigators may wish to consider using the primary antibody conjugated to sepharose or magnetic beads as an alternative to using secondary immunoprecipitation reagents (e.g., Protein A-Agarose) as described here. Combining primary antibodies directed to different epitopes of the same protein may be advantageous in some cases.

Protocol tips
-Investigators may wish to consider using the primary antibody conjugated to sepharose or magnetic beads as an alternative to using secondary immunoprecipitation reagents (e.g., Protein A-Agarose) as described here. Combining primary antibodies directed to different epitopes of the same protein may be advantageous in some cases.

Publication protocol

A ChIP assay was performed using an Imprint Chromatin Immunoprecipitation Kit (Sigma, St. Louis, MO, USA) according to the manufactures protocol. Briefly, MCF-7 cells were fixed with 1 % formaldehyde and quenched with 1.25 M glycine. After washing with PBS, the cells were suspended in nuclei preparation buffer and shearing buffer, and sonicated under optimized conditions. This sheared DNA was then centrifuged and a cleared supernatant was used for protein/DNA immunoprecipitation. The clarified supernatant was diluted with dilution buffer (1:1 ratio) and 5 μl of diluted samples were removed as an internal control. The diluted supernatant was incubated with antibody (STAT5b) in pre-coated wells for 90 min. For negative and positive control, normal mouse IgG and anti-RNA polymerase II were used, respectively. The unbound DNA was washed off with IP wash buffer and the bound DNA was collected by cross link reversal using DNA release buffer containing proteinase K. The released DNAs and the DNA from the internal controls were purified with GenElute Binding Column G. The DNA was then quantified using conventional PCR.

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