TransAM® ER Transcription Factor ELISA Kits

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mix gently avoid foaming during the dilution of Antibody Binding Buffer. -The developing solution should be warmed to room temperature at least for 1 hour before use. - Keep away from light as the developing solution is light sensitive.	-Discard diluted antibody buffer after use. -After use, discard remaining developing solution.

Protocol tips
-Mix gently avoid foaming during the dilution of Antibody Binding Buffer. -The developing solution should be warmed to room temperature at least for 1 hour before use. - Keep away from light as the developing solution is light sensitive.
Downstream tips
-Discard diluted antibody buffer after use. -After use, discard remaining developing solution.

Publication protocol

To test the binding capabilities of ERα to the putative EREs containing either the C or G allele of rs2267735, we performed a competitive binding enzyme-linked immunosorbent assay using the TransAM ER kit (catalog no. 41396; Active Motif, Carlsbad, CA, USA). To each microwell coated with canonical ERE sequence oligonucleotide (oligo), we added estradiol-treated MCF-7 nuclear extract (5 μg; catalog no. 36016, Active Motif) and 10–100 pmol of one of four double-stranded oligo DNAs (two competing/experimental sequences plus a negative and positive control). The sequences of the oligos used for this experiment are provided in Supplementary Table 1. Each experimental assay was performed in triplicate. The controls were performed in duplicate. Individual oligos and nuclear extract were added to each microwell and incubated for 1 h. Next, a primary antibody (ERα 1:1000) and then secondary antibody (horseradish peroxidase-conjugated IgG; 1:1000) were added for 1 h each. After several rinses, a colorimetric reaction to horseradish peroxidase was initiated and absorbance was measured at a wavelength of 450 nm to determine binding efficiencies. Antibodies and other reagents were provided in the TransAM ER kit.

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Papers

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Manufacturer protocol

Download the product protocol from Active Motif for TransAM® ER Transcription Factor ELISA Kits below.

Download manufacturer protocol

Videos

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