Two-dimensional pulsed electrophoresis

Question 25 PCR Isoelectric focusing two-dimensional gel electrophoresis capillary gel electrophoresis and pulsed-field gel electrophoresis... 

Two-Dimensional Pulsed Field Electrophoresis Theoretical Background. Applying the polymer reptation model to conventional DNA electrophoresis is proving to be quite successful, especially in the case of field strength dependent electrophoretic mobilities. 

A combination of circular dichroism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical crosslinking, and analytical ultracentrifugation studies showed that both the apo- and metallated derivatives of H21(31-mer) form two-stranded a-helical coiled coils in aqueous solution. Further characterization of these derivatives by EPR spin-label experiments helped to determine its three-dimensional backbone structure. In these studies, a Cys-21 mutant of the 31-mer coiled coil, H21/C21(31-mer), was prepared and labeled with a thiol-specific nltroxide spin label (MTSL = l-oxyl-2,2,5,5-tetramethyl-A -pyrroline-3-methyl-methanethiosulfonate) at position 21 of the peptide sequence which is the site of metal substitution in the ET heterodimer. Comparison of the low-temperature, dipolar-broadened spectrum of the spin-labeled dimer with those of magnetically dilute peptide samples yielded a backbone-to-backbone distance that was nearly identical to that of the GCN4 homodimer. Based on these results, computer modeling studies provided an estimate of the metal-to-metal distance in the ET heterodimer of m-m > 25 A. The electron-transfo properties of this system are now being studied by a combination of laser flash-quench and pulse radiolysis techniques. 

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