Program ElphoFit 2.3

Dietmar.Tietz at agrar.uni-giessen.de Dietmar.Tietz at agrar.uni-giessen.de
Tue Jul 12 08:41:10 EST 1994


Program ElphoFit 2.3
====================

Software for evaluating data derived from native gel electrophoresis 
of proteins, DNA, viruses, vesicles, conjugated vaccines etc.  
Analysis is based on linear or nonlinear Ferguson plots 
[plot of log(mobility) vs. gel concentration].  

Mobility = migration velocity (cm/s) / field strength (V/cm)

Calculations use a physical model rather than empirical polynomial 
functions.  A Newton-Gauss Marquardt-Levenberg curve fitting 
algorithm is employed.  The Program is designed for scientific 
purposes; it was not the aim to create a 100% Macintosh-like interface.  
Requirements: Apple Macintosh II and Quadra families; Systems 6. or 7, 
up to 256 colors or gray levels; 32 bit addressing off is the preferred 
mode.


Questions ?  Suggestions ?  
E-mail:   Dietmar.Tietz at uni-giessen.de

ElphoFit 2.3 is freeware and was developed at the NIH, Bethesda, 
Maryland.  It is available on PCBull (NIH) and America Online. 
If you do not have access to these networks, please mail a Macintosh-
formatted 3.5" floppy disk AND the equivalent of US$5.00 (cash or 
USA personal check) for overseas postage and handling.  In the EC,
please enclose the equivalent of DM3.00.

ADDRESS:

Dietmar Tietz
Biostatistics
Justus-Liebig-University
Ludwigstr. 27
D-35394 Giessen, Germany

ElphoFit 2.3 is user-friendly and provides lots of helpful comments.
But it does not come with an extensive documentation.  Additional
information (theory, rationales, features, data in/output and graphics) 
is provided in the literature listed below.  I will be happy to answer 
further questions by Email.


REFERENCES (provided upon request):

AU  - Tietz D
AU  - Chrambach A
TI  - DNA shape and separation efficiency in polymer media: a
computerized method based on electrophoretic mobility data.
AB  - The computer program ELPHOFIT for evaluation of the nonlinear
plots of log-(mobility) vs. polymer concentration (Ferguson
plots) in terms of molecular and polymer properties has been
extended to yield a measure of the molecular sieving capacity of
he polymer medium. The usefulness of the extended program,
version 2.2, was exemplified by the evaluation of DNA shape and
separation efficiency in solutions and gels of agarose and
polyacrylamide, using previous reports in the literature as a
data base. That application of the extended program yields the
following results:(i) The size of migrating DNA can be compared
with an equivalent sphere having the same free mobility for a
particular set of experimental conditions. The decrease in size
of the equivalent sphere with polymer concentration previously
demonstrated for agarose solutions applies to all of these
polymer media; it reveals a steep, hyperbolic decline of that
radius in uncrosslinked polyacrylamide solutions in contrast to
he shallow decline in the other three media. (ii) The separation
efficiency of polyacrylamide gels exceeds that of uncrosslinked
polyacrylamide solutions; the separation efficiency of agarose
solutions for DNA smaller than 1 kb in length is higher than that
of polyacrylamide solutions. Program ELPHOFIT 2.2 is available on
request from the first author.
AD  - Section on Macromolecular Analysis, National Institute of Child
Health and Human Development, National Institutes of Health,
Bethesda, MD.
SO  - Electrophoresis 1993 Mar;14(3):185-90

AU  - Tietz D
TI  - Analysis of one-dimensional gels and two-dimensional Serwer-type
gels on the basis of the extended Ogston model using personal
computers.
AB  - This report presents the stand-alone computer application
ELPHOFIT, a software package for the analysis of gel
electrophoretic data based on Ferguson plots. Either conventional
one-dimensional gels or two-dimensional agarose gels
(Serwer-type) can be evaluated. Special emphasis is on the latter
gel type, which has been applied previously for the separation of
DNA, intact viruses and polydisperse meningitis vaccines.
ELPHOFIT is designed for Macintosh PCs and for the IBM XT, AT,
PS/2 and compatibles. The program operates interactively with the
user, who determines the course of evaluation. Data input is in
the format of files providing values of gel electrophoretic
migration distances or particle mobility (absolute or relative).
Data processing involves a simultaneous least-square curve
fitting algorithm (Newton-Gauss, Marquardt-Levenberg) which uses
equations derived from the extended Ogston model. Functions are
fit to the database by adjusting their variables, representing
physical parameters of the gel and the electrophoresed particle.
The program output consists of tables and graphics accompanied by
an explanatory text providing the following information: (i)
radius and free mobility of the electrophoresed particle, (ii)
fiber radius, length and volume, mean or median pore radius of
the gel, (iii) linear Ferguson plots, (iv)
iso-free-mobility/iso-size nomogram for two-dimensional gels, (v)
confidence ellipses, (vi) required parameters for image
processing program GELFIT and (vii) goodness-of-fit and other
statistical parameters, such as standard errors, dependency
values, root-mean-square (RMS) error and determination
coefficient. Other features of the program are (i) simulation of
Serwer-type two-dimensional electrophoresis, (ii) standardization
according to size, or size and free mobility, (iii) the
conversion of particle radii to molecular (or particle) weight
and vice versa, (iv) interconversion of DNA size specifications,
i.e. the number of base pairs and the geometric mean radii, (v)
computation of gel concentration for optimal resolution of two
components, (vi) option to obtain a session record, (viii) option
to establish a data output file containing the information of
generated graphics (IBM only) and (ix) a text editing facility,
e.g., for creating data files. Graphics (Macintosh version, PICT
format) and text output files (both IBM and Macintosh versions,
standard ASCII format) generated by ELPHOFIT are compatible with
commercially available software.
AD  - Laboratory of Theoretical and Physical Biology, National
Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, MD 20892.
SO  - Electrophoresis 1991 Jan;12(1):28-39

AU  - Tietz D
AU  - Aldroubi A
AU  - Schneerson R
AU  - Unser M
AU  - Chrambach A
TI  - The distribution of particles characterized by size and free
mobility within polydisperse populations of
protein-polysaccharide conjugates, determined from
two-dimensional agarose electropherograms.
AB  - New approaches for the characterization of polydisperse particle
populations are presented*. The investigated samples contain
virus-sized protein-polysaccharide conjugates which had
previously been prepared as immunogens against bacterial
meningitis (Hib). The analysis is based on two-dimensional
agarose electrophoresis (Serwer-type). This method, like the one
of O'Farrell, achieves a separation according to size and charge.
It relies on a different principle, however, and is applicable to
nondenatured particles which are 100 to more than 1000 times
larger in mass than regular uncrosslinked proteins. Data from
stained gel patterns are evaluated by the computer program
ELPHOFIT, which makes it possible to standardize the gel and to
construct a nomogram which defines every position on the gel in
terms of particle size and free mobility (related to surface net
charge density). The output of ELPHOFIT, consisting of nomogram
parameters, is transferred to the image processing program
GELFIT. This software is used to evaluate the computer images
obtained by digitizing the stained gel patterns: (i) The nomogram
is electronically superimposed on the computer image. (ii) The
gel pattern is transformed from a curvilinear to a rectangular
coordinate system of particle size and free mobility. The center
of gravity as well as density maxima are given in coordinates of
particle size and free mobility. Ranges of grey levels can be
accentuated by adding 16 pseudocolors. (iii) Using
surface-stripping techniques, GELFIT provides an estimate for the
number of major subpopulations within each preparation. (iv)
Numerical values for the distribution of particle size and free
mobility are determined. Using program IMAGE, the quantitative
physical assessment of a given conjugate preparation is presented
in the form of a computer-generated three-dimensional plot, the
shape of which serves to identify and characterize the
preparation visually. The data analysis based on digitized
two-dimensional gel patterns is automated to an extent that a
technician can perform routine evaluations. It uses the Macintosh
II personal computer.
AD  - Laboratory of Theoretical and Physical Biology, National
Institute of Child Health and Human Development, NIH, Bethesda,
MD 20892.
SO  - Electrophoresis 1991 Jan;12(1):46-54

AU  - Tietz D
AU  - Chrambach A
TI  - Concave Ferguson plots of DNA fragments and convex Ferguson 
plots of bacteriophages: evaluation of molecular and fiber properties,
using desktop computers.
AB  - A desktop computer program evaluating physical properties of 
DNA and bacteriophages is presented. The analysis is based on data
obtained from capillary and submarine-type agarose
electrophoresis. Native molecular/particle properties and
properties of the gel (or polymer) medium can be derived from
electrophoresis at several gel concentrations. This is done
conveniently by a computerized evaluation of the semi-logarithmic
plot of mobility vs. gel concentration, designated the Ferguson
plot. In application to most proteins, this plot is linear and
computer programs exist to evaluate it. However, nonlinear
Ferguson plots have assumed great importance in view of the fact
that the plots are concave for DNA. Similarly, convex plots are
important since they prevail in the electrophoresis of large
particles in agarose. The computer program reported here is the
first to (i) address concave Ferguson plots and (ii) allow for
the evaluation of both cases using a desktop computer. Program
ELPHOFIT version 2.0, a Macintosh application, is available upon
request.
AD  - Section on Macromolecular Analysis, National Institute of Child
Health and Human Development, National Institutes of Health,
Bethesda, MD 20892-0001.
SO  - Electrophoresis 1992 May;13(5):286-94

AU  - Orban L
AU  - Garner MM
AU  - Wheeler D
AU  - Tietz D
AU  - Chrambach A
TI  - Characterization of the electrophoretic properties of 
nucleosome core particles by transverse polyacrylamide pore
gradient gel electrophoresis.
AB  - Transverse pore gradient gel electrophoresis, previously applied
to bent DNA, has extended the usefulness of the gel retardation
assay in two ways: (i) by differentiating between different DNA
conformations; (ii) by providing information regarding the
physical properties of DNA. In the present study, similarly
extended information is obtained with regard to a
well-characterized DNA-protein complex, the chicken erythrocyte
nucleosome core particle. (i) The winding of DNA around the
protein core constrains the DNA which renders its Ferguson curve
(migration distance vs. gel concentration) similar to that of
kinetoplast DNA, i.e. it intersects sharply with the Ferguson
curves of linear DNA standards. By contrast, the deproteinized
nucleosome DNA exhibits a Ferguson curve similar to linear
standards of the same length. (ii) Interpretation of the Ferguson
curve based on a mathematical model shows that the nucleosome
exhibits a linear Ferguson plot [log(mobility) vs. gel
concentration]. This is similar to and characteristic of
spherical proteins, contrasting with the concave plot typical for
linear and bent DNA. (iii) The effective size of the nucleosome,
evaluated in terms of an equivalent sphere (i.e. a hypothetical
spherical particle with a radius, Res, having the same
electrophoretic mobility as DNA for a particular set of
experimental conditions), remains invariant across the gel
concentration range of 3-9%T. This is similar to proteins and
bacteriophages and contrasts with the progressive decline of Res
with increasing gel concentration observed for linear DNA and the
deproteinized nucleosomal DNA.
AD  - Section on Macromolecular Analysis, National Institute of Child
Health and Human Development, National Institutes of Health,
Bethesda, MD 20892-0001.
SO  - Electrophoresis 1993 Aug;14(8):720-4

Tietz, D.:  Evaluation of mobility data obtained from gel 
electrophoresis:  Strategies in the computation of particle and gel 
properties on the basis of the extended Ogston model  (Review). 
Advances in Electrophoresis, Vol. 2, 109-169 (1988). 






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