Gel Retardation Assays for DNA-binding Proteins

Abstract

MATERIALS 10x Tris-glycine buffer TBE buffer may be used in place of Tris-glycine. 6x Gel-loading buffer I 32P-labeled control DNA 32P-labeled target DNA of >20 bp (specific activity 2 x 107 cpm/μg [ Labeling of the DNA fragment can be accomplished by phosphorylation (Dephosphorylation of DNA Fragments with Alkaline Phosphataseor Phosphorylating the 5' Termini of Oligonucleotides), filling in of a 3'-recessed end using a DNA polymerase (Labeling 3' Termini of Double-stranded DNA Using the Klenow Fragment of E. coli DNA Polymerase Ior Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I), or by using PCR to incorporate radiolabeled nucleotides into the body of the probe (The Basic Polymerase Chain Reaction). Control nuclear extract or protein fraction Ficoll 400 (20% w/v) Dissolve the Ficoll in sterile H2O and store the solution frozen in 100-μl aliquots at -20°C. Nuclear extract or Protein fraction(s) Prepare the extract by one of the methods described in Mapping Protein-binding Sites on DNA by DNase I Footprinting. Poly(dI-dC) (1 mg/ml) Gel Retardation Assays for DNA-binding Proteins -Sambrook and Rus... file:///C:/Users/MILI%20JOON/Documents/protocol/Electrophoresis,...