Determination of the molecular weight of DNA-bound protein(s) responsible for gel electrophoretic mobility shift of linear DNA fragments examplified with purified viral myb protein.
A protein-DNA complex has less gel electrophoretic mobility than the free DNA fragment. One parameter for the degree of retardation of a linear DNA fragment in a protein-DNA complex is the molecular weight of the bound protein(s). The quotient of the migration distances of free DNA (m) and protein-DNA complex (m') is a function of the molecular weight (MW) of the bound protein(s). Based on the evaluation of the lac repressor induced mobility shift of a 203 bp DNA fragment containing the lac operator in a 5% non-denaturating polyacrylamide gel a direct proportionality could be shown between (m/m'-1) and MW with the proportionality factor K = 215 kDa. The factor K depends on the acrylamide concentration in the gel, getting lower values with increasing acrylamide concentrations. A calculation is given to determine the molecular weight of DNA-binding factors responsible for the decreased electrophoretic mobility of a linear DNA fragment. As an example this calculation was used in order to analyse DNA-binding of the isolated viral myb protein. It could be demonstrated that the viral myb protein binds to DNA as a monomer and as a dimer.
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Max-Planck-Institut für Molekulare Genetik, Abt.Schuster, Berlin, FRG.
D009189:Avian Myeloblastosis Virus / Q000378:metabolism; D010582:Bacteriophage lambda / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D020626:Oncogene Proteins v-myb; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism; D012191:Retroviridae Proteins / Q000378:metabolism*
D004279:DNA, Viral; D020626:Oncogene Proteins v-myb; D012097:Repressor Proteins; D012191:Retroviridae Proteins