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This chapter describes three complementary footprinting methods and the protocols used for analysis of polyamide : DNA complexes: (1) MPE Fe(II) footprinting, (2) affinity cleavage, and (3) quantitative DNase I footprint titration.
The DNase I footprint locations were from http://noble.gs.washington.edu/proj/footprinting/yeast.footprints.bed [20].
To explore the exact binding regions of the NAPs we performed DNase I footprint experiments with the individual DNA-protein complexes.
To identify possible candidates, we used Patser to scan HSF-bound promoters and introns for matches (Bonferroni corrected p-value<5.6×10−2) to PWMs representing 111 different DNA binding proteins from two databases, Transfac and the Drosophila DNase I Footprint Database.
We repeated these steps to identify matches to all other unique PWMs from TRANSFAC and from the Drosophila DNase I Footprint Database this time counting matches to each matrix in HSF-bound promoters and HSF-bound introns with Bonferroni corrected p-values <5.6×10−2.
(D ) In vitro DNase I footprint assay to identify the SerRS binding site.
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In pulmonary fibroblasts (IMR90), significantly low methylation was detected at CpG dinucleotides within DNase I footprints, compared to CpGs in non-footprinted regions of the same DNase I hypersensitive site (DHS).
(D) DNase I footprinting.
DNase I footprinting revealed extensive contacts between PHO/PCC and the PRE.
Quantitative DNase I footprinting analysis indicates that perfect sites bind PA1 with Kds between 0.7 and 2.2 nM.
The DNA binding properties of diamino polyamide 5 were determined using a number of techniques including CD, ΔTM, DNase I footprinting, SPR and ITC studies.
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