Transverse Pore Gradient Electrophoresis (TPGE)

(Contributor: Andreas Chrambach, National Institutes of Health (NIH), Bethesda, MD)

Introduction

Gel electrophoresis carried out in a gel formed to contain a gradient of pore size, of solvent, of detergent, of urea or of other additive, and oriented orthogonally to the direction of gradient formation, is designated as Transverse Pore Gradient Electrophoresis (TPGE).

Operationally, the transverse pore gradient gel is most easily applied by turning a commercial pore gradient gel at a right angle in a horizontal gel electrophoresis apparatus and covering the gel surface with the sample. Commercially unavailable pore size gradients and all other forms of gradient gels must be formed and gelled in the laboratory, usually by means of a 2-chamber gradient maker.

The unique function of transverse gradient gels is that they pinpoint critical transitions in conformation and/or size of a molecular species as a function of the concentration of the particular additive applied (1). Moreover, pore size gradient gel electrophoresis provides an experimental gel pattern of migration distance vs gel concentration, i.e. qualitatively a non-logarithmic Ferguson plot (designated as a Ferguson curve) (2).

The relation is qualitative unless the pore size gradient is perfectly linear which, in practice, it is not. Nonetheless, the gel pattern reveals on inspection the gel concentration range for the optimal separation of any two species. It should be noted that thereby optimal separation conditions can be established in about 1 hour, using PhastSystem (Pharmacia) minigels (3), while obtaining the identical information by Ferguson plot analysis would require an entire workday.

Applicability

  1. Transverse pore gradient gels provide unequivocal information as to the number of components even with mixtures of 30 to 50 components that cannot be determined by Ferguson plot analysis using gels of various concentrations.
  2. Finally, the shapes of the Ferguson curves allow one to discriminate between species of the same conformational type (non-overlapping Ferguson curves) and those of a different conformation (intersecting Ferguson curves) in application to molecules of equal surface charge density such a polynucleotides (4).
  3. The information residing in the Ferguson curves can be quantitatively evaluated by image analysis (2), provided that the shape of the gradient has been determined.

Literature References

  1. Creighton, T. E.: Enzyme Structure, Part L (Hirs, C.H.W., Serge N. Timasheff, S.N., eds.) Methods Enzymol. 131: pp.156-172 (1986)
  2. Wheeler, D., Orban, L. Garner, M. M. and Chrambach, A.: Computer-Aided Analysis of DNA Curves on Transverse Gradient Gels. J.Biochem.Biophys.Methods 24: pp.171-180 (1992)
  3. Buzas, Zs., Wheeler, D. L., Garner, M. M., Tietz, D., Chrambach, A.: Transverse pore gradient gel electrophoresis, using the PhastSystem. Electrophoresis 15: pp.1028-1031 (1994)
  4. Wheeler, D., Lin, Jia-Hwei and Chrambach, A.: Distinction between Supercoiled and Linear DNA in Transverse Agarose Pore Gradient Gel Electrophoresis. Electrophoresis 13: pp.403-406 (1992).