Smart-DNA-Pull™ DNA Pull-down Kit

Overview

Description

DNA-Protein Pull-down

Product Picture

Applications

Highlights

1. This kit is suitable for adherent cells, suspended cells, animal tissues, plant tissues, and purified recombinant proteins. It is suitable for various lengths and complexities of DNA probes labeled with terminal biotin. Proteins that are endogenous, overexpressed, or translated in vitro are suitable as sample types.


2. This kit uses biotin labeled DNA probes to directly capture target proteins, such as transcription factors. No antibodies are required for pull down, and magnetic bead method is used without centrifugation. After completing the DNA labeling reaction, very little manual operation time is required (less than 3 hours).


3. This reagent kit is suitable for the interaction between DNA sequences and DNA binding proteins such as transcription factors, transcription cofactors, DNA damage repair factors, etc. It can be used for downstream Western blot verification detection of known proteins and mass spectrometry identification of unknown proteins.


4. This manual is a comprehensive technical manual for DNA Pull down experiments, summarizing detailed information on the interactions between DNA and various proteins. After reading this nanny level manual, all difficulties will be easily solved, and DNA Pull down is within your grasp.

Work Flow Image

Results Display

DNA Pull-Down and WB assay
 

Ncoa3 is recruited via Esrrb to ERREs at target loci. (A) DNA pull-down assays using previously described wild-type (Wt) or ERRE mutated sequences from Esrrb, Klf4, Sox2 (Feng et al. 2009), or Nanog (van den Berg et al. 2008). Biotinylated probes (40–50 base pairs [bp]) were incubated with extracts from COS-1 cells transfected with Flag-Esrrb and Ncoa3 and recovered on streptavidin beads, and DNA-associated proteins were visualized by Western blotting. (B) Esrrb and Ncoa3 enrichment levels at Esrrb, Klf4, Nanog, and Sox2 ERREs and an intergenic (Inter.) control region in ESCs as assessed by ChIP and qPCR and expressed relative to input. Data are the mean 6 SEM of three biological replicates. (C) Ncoa3 and Esrrb enrichment levels at candidate loci following Esrrb depletion in ESCs, expressed relative to input. Enrichment for shGFP transfected cells is set at 100% in each individual experiment. Data are the mean 6 SD of at least two independent experiments. Dotted lines in B and C indicate background enrichment by control IgGs (Santa Cruz Biotechnology). (D) Western blotting showing specific Esrrb protein depletion 48 h after transfection with shEsrrb. Note that Ncoa3 and Oct4 levels are unchanged at this time. (E) DNA pull-down assays using Nanog wild-type or ERRE mutated probe, which also contains a neighboring Oct–Sox site (van den Berg et al. 2008), and cell extracts from COS-1 transfected with Ncoa3, Oct4, and Sox2.