biomedical research biomedical research biomedical research biomedical research biomedical research biomedical
research plasmid DNA plasmid DNA plasmid DNA plasmid DNA plasmid DNA plasmid DNA adenovirus adenovirus
adenovirus adenovirus adenovirus adenovirus adenovirus lactate lactate lactate lactate lactate lactate lactate lactate
chemiluminescent chemiluminescent chemiluminescent chemiluminescent chemiluminescent TMB TMB TMB
chemiluminescent TMB TMB TMB TMB TMB TMB TMB genomic genomic genomic genomic genomic genomic RNA
RNA RNA RNA RNA RNA RNA RNA western blotting western blotting western blotting western blotting protein assay
protein assay protein assay protein assay protein assay SDS-PAGE SDS-PAGE SDS-PAGE SDS-PAGE SDS-PAGE
luciferase luciferase luciferase luciferase luciferase luciferase luciferase MTT MTT MTT MTT MTT MTT MTT LDH
LDH LDH LDH LDH LDH LDH cell injury cell injury cell injury cell injury cell injury cell proliferation cell proliferation
galactosidase galactosidase galactosidase galactosidase galactosidase galactosidase competent cell competent cell
competent cell competent cell biomedical research service biomedical research service biomedical research
Differential detection of multiple DNA-binding complexes using dissimilar polyanion competitors

Although gel shift (or gel retardation) assay is routinely used to detect DNA-binding activity, the assay when performed with crude protein extracts requires the use
of non-specific DNA competitors to sequester undesired DNA-protein interactions. These DNA competitors are typically polyanionic polymers, the most commonly
used of which is poly(dI-dC). However, since DNA-binding proteins vary widely in their nucleic acid binding affinity and specificity, the use of poly(dI-dC) alone for
identification of a novel DNA-binding activity present in crude extracts can be problematic and challenging. In theory, it is of benefit to use a set of structurally
dissimilar polyanion competitors to achieve the goal. In addition, salt concentrations in the binding buffer may differentially affect DNA-protein and protein-protein
interactions (Mol. Cell. Biol. 11:5090,1991).

We have indeed found that differential use of dissimilar polyanion competitors and variation of salt concentrations can facilitate detection of multiple specific
DNA-binding protein complexes present in crude protein extracts. For instance, the ~200-bp avian skeletal alpha-actin proximal promoter contains binding sites for
two major transcription factors, YY1 and SRF. Notably, gel shift assays using crude muscle extracts and poly(dI-dC) only detected the YY1-DNA complex (Nucl. Acid
Res. 20:140,1992). The use of poly(dG-dC) or salmon sperm DNA in gel shift assay, however, readily picked up the SRF-DNA complex from the crude muscle
extracts. Using crude muscle protein extracts, we were able to demonstrate competitive binding of YY1 and SRF to the alpha-actin proximal promoter and illustrate
an interactive gene regulatory mechanism governing the expression of muscle-restricted genes during myogenesis (Proc. Natl. Acad. Sci. USA 89:9814, 1992).  

We note that the heteropolymer poly(dG-dC) has been used to detect a family of TATA box-binding factors (Cell 66:563, 1991). The DNA element bound by SRF
consists of an AT-rich inner sequence, the recognition of which by SRF may be preferentially weakened by poly(dI-dC) but not by poly(dG-dC) or salmon sperm
DNA. Our
Gel Shift Assay Kit thus includes a set of three different polyanion polymers: poly(dI-dC), poly(dG-dC), and sheared salmon sperm DNA. The binding
assay can be performed in two different salt concentrations (10 and 100 mM KCl) to facilitate the detection of different types of higher-order DNA-protein
complexes.  
Site Map
Biomedical Research Service
& Clinical Application
Alphabetical Listing