Journal: Molecular Oncology
Article Title: High affinity binding of H3K14ac through collaboration of bromodomains 2, 4 and 5 is critical for the molecular and tumor suppressor functions of PBRM1
Figure Lengend Snippet: Quantitative measurement confirms that BD 245 work together to create high affinity to H3K14ac. (A) The indicated GST ‐ BD constructs of PBRM 1 were expressed and purified from Escherichia coli , pulled down by the indicated peptides, and immunoblotted with anti‐ GST antibody. (B) The bands in (A) were quantified with NIH ImageJ, and the ratios of bands associated with H3K14ac/H3 were calculated from three experiments. The P ‐values were calculated using the two‐tailed Student's t ‐test. *: P
Article Snippet: 2.2 Protein expression and purification Polybromo‐1 constructs consisting of BD2, BD4, BD234, and BD245 were subcloned into pGST parallel expression vectors (Sheffield et al ., ) individually expressed in Escherichia coli strain BL21, purified over a GST column (GE Healthcare, Pittsburgh, PA, USA; Catalog #17‐5130‐01), eluted with 10 mm reduced glutathione, and dialyzed against 2 L of dialysis buffer (25 mm Tris/HCl, 500 mm NaCl, pH 8.0) to remove glutathione.
Techniques: Construct, Purification, Two Tailed Test