TFIIB Antibody (D-3): sc-271736

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

ChIP was performed using the Magnify Chromatin Immunoprecipitation System (Invitrogen), according to the manufacturer’s instructions. Cells transfected with PC4-Flag or Flag empty vector, treated with or without TSA, were collected and washed with 1x PBS, and then fixed with 1% formaldehyde for 10 min. Chromatin was then prepared by sonication shearing, optimized according to the manufacturer’s instructions. ChIP was performed on sheared chromatin from approximately 2 × 105 cells, using IgG antibody as a negative control or antibodies recognizing Flag, TFIIB, Pol II, H3K9-Ac, or H3.3. Chromatin purified from an equivalent number of cells not subjected to the immunoprecipitation step was used as the input control. Real-time PCR was performed on DNA isolated from each of the ChIP reactions using primer pairs specific for LHR 5′-ACTGGGCACTGTCGCAGGTC-3′ (sense) and 5′-CATGGCCGGCGAACTGGGCT-3′ (anti-sense) [6]. The amplified region of the 189 bp LHR promoter contains the relevant proximal Sp1 response element.

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Manufacturer protocol

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