PMC:4996381 / 46546-48029 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996381","sourcedb":"PMC","sourceid":"4996381","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996381","text":"Figure 2 Comparison of BRD4(1) binding to peptides using SPOT arrays and BLI. Membranes carrying histone H2A, H2B, H3 and H4 15-amino acid long peptides with single or multiple lysine acetylation modifications were prepared on cellulose support. Peptide density was controlled using a ratio of Fmoc-β-Alanine to Ac-β-Alanine that ranged from 1:0 to 1:10 in the first step of synthesis, effectively reducing the amount of peptide found on each SPOT of the array. His6 tagged BRD4(1) was used to identify binding after incubating membranes overnight at 4 °C. Bound protein was detected using an anti-his antibody (His-tag® Antibody HPR conjugated, Novagen, #71841). SPOT intensity was measured on a luminescent image analyser (Luminescent Image Analyser LAS-4000 Fujifilm) using the KODAC 1D software package (Kodak 1D Scientific Imaging System V.3.6.2.). His8 peptides were used as controls for antibody binding and array intensities were normalized between 0 and 100 using the control peptide intensity. The last column on the graphs depicts binding of BRD4(1) to the same peptides derived from a commercial set (AltaBioSciences Histone array, Set 4 Histone Acetyl-Lysine library) carrying biotinylated histone peptides used in a biolayer interferometry experiment (BLI). Binding was normalized for this experiment and the scale is given in the inset. Strongly bound peptides showed binding in both methods while weaker binding peptides were over-represented in the peptide array. ","tracks":[]}