Iranian Biomedical Journal

Running head: ELECTROPHORETIC MOBILITY SHIFT ASSAY 1
Electrophoretic mobility shift assay using liver nuclear extract
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ELECTROPHORETIC MOBILITY SHIFT ASSAY 2
Electrophoretic mobility shift assay using liver nuclear extract
The electrophoretic mobility shift assay is a biochemical procedure that elucidates binding
between proteins and nucleic acids by detecting a shift in bands in gel electrophoresis.The
strand (Schwanhäusser & Busse et al. , 2011). EMSA uses a polyacrylamide gel that causes a
caging effect that maintains the high local concentration of the components and effectively
sets the equilibrium of the biomolecular recombination reaction towards complex molecule
formation.The EMSA technique enables the DNA fragments carrying bound proteins to be
separated from uncomplexed DNA.EMSA separates biomolecules based on weight (Meshkani
& Pasalar at el ., 2013)
Information on the EMSA of liver nuclear extract using C-site oligo primers will be used to
answr the fololwing questions.
Questions
1. Calculate the volumes needed to produce the final concentarions of 1x binding
buffer,1μgpoly [d-(1-c)], 0.155 pmol DIG-C-site,15.4 pmol unlabelled C-site
oligo,15,4 pmol unrelated (GAL-4) oligo,1μgHepG2 nuclear exctract in a 20μl
final volume for each reaction mixture.Stock solution concentration are given in
the brackets next to reagents. (2marks)
Calculation of the final volumes and final amounts of reagents.
Volumes of reagents are calculated by C1V1=C2V2 formula as follows:C1 =initial
concetration,V1;initial volume,C2;final concentarion and v2;final volume. Thus,
C1=5,C2=1,V2 =20 and V1=?
Binding buffer: 5×v1=20×1;v1=4μl