>> On 15 Jul 1994 15:14:16 GMT, Stephen R. Lasky wrote:
>> (some stuff deleted)
>> >In general though, we use denaturation in the presence of SDS so that a
> >protein will migrate at a rate relative to its mass and not be effected by
> >amino acid composition.
>> That's the generally accepted therory and, no doubt, many if not most
> proteins fit nicely into that scheme. But there are exceptions, just one
> example (might be interesting for Jean-Marc and Emma): There is a protein
> called dTAF40 because on a SDS gel it has the apparent molecular mass of 40
> kd. Molecular cloning of the cDNA revealed a molecular mass of only 29 kd
> (Cell 75, 519-530, 1993). Could anybody kindly give an explanation for this
> unusual migration on a SDS gel?
>> Matthias Zeiner
> Inst. Biol. Chem.
> Heidelberg
>>Dear Mathias Zeiner:
Anomalous migration in SDS electrophoresis usually means
that the treatment with SDS DOES NOT produce a uniform surface
net charge density. It happens more often than expected, just it is
not recognized, since it cannot be detected on a SINGLE gel.
What is needed in such a case is the so-called Ferguson plot
technique. It also allows for characterizing native proteins without
SDS treatment. Please find attached some more information
about Ferguson plots. Please feel free to ask me more
questions.
Best regards, Dietmar Tietz
The rationales of Ferguson plot analysis
========================================
Gel electrophoresis is usually performed at one single gel concentration.
Migration distances of sample zones are compared with the position of
standards and the molecular size of unknowns is determined.
Gel electrophoresis depends on both particle size and surface net charge
density. Determining particle sizes in ONE gel will only work, if the
sample and standards have the SAME charge. This is usually true for
nucleic acids and SDS denatured proteins. If not so, the "single gel -
one lane of standards method" can be compared with solving one
equation (migration distance) with two unknowns (size and charge).
Depending on the setting, one can determine almost any size that is
desired. "House numbers" are also estimated, if sample and standards
are of different nature, i.e., vary in conformation. This phenomenon is
called anomalous migration or peak inversion in the literature, but it is
nothing else than comparing, e.g., apples and bananas.
Often it is useful to exploit differences in both charge and size. Or one
would like to investigate native samples, such as intact viruses, vesicles,
etc. Or we know that the conformation varies, i.e., that we have linear
and circular DNA. In all these cases we can apply the Ferguson plot
method which allows to distinguish different particle shapes, sizes and
surface net charge densities at the same time.
Since we ask for more information, it is necessary to provide more
input data. We have to study the migration of particles not at one, but
SEVERAL gel concentrations. Particle migration is measured as
mobility.
Mobility = migration velocity (cm/s) / field strength (V/cm).
Then we plot log(mobility) vs. gel concentration (Ferguson plot). This
plot will be linear or nonlinear depending on the problem at hand. The
plot may be evaluated by program ElphoFit. It uses file input.
The output consists of graphics and tables providing statistical
information. Part of the output are the size and free mobility (mobility
in the absence of a gel, related to surface net charge density) of
unknown particles. The size is determined in terms of effective
molecular radii. The program allows to convert these radii to values
of molecular (or particle) weight in Dalton or base pairs (bp).
Parameters descriptive of the gel matrix are also specified.
The slope of linear Ferguson plots is proportional to particle size. The
intercept on the ordinate specifies particle free mobility. Particles of
similar size have parallel Ferguson plots. Particles with the same
surface net charge density have the same intercept on the log(mobility)
axis (ordinate). This way, charge and size isomers can easily be
detected.
Non-spherical particles produce nonlinear Ferguson plots. Nonlinearity
may also occur, if gel matrix parameters vary with gel concentration.
The slope of nonlinear plots close to 0 gel concentration is related to
particle size. Size estimates are usually pretty good, even if differently
shaped Ferguson plots are compared that originate from particles of
different conformation.
Summarizing: The Ferguson plot method is more laborious, but yields
more information and is capable of detecting anomalies which would
remain undetected on a SINGLE gel. Sharp bands on a single gel do not
warrant correct results.
Program ElphoFit 2.31
=====================
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] which are explained
in the File "Why Ferguson plots?".
Calculations use a physical model rather than empirical polynomial
functions. A Newton-Gauss Marquardt-Levenberg curve-fitting
algorithm and a mathematical approach based on the "random space
walk of a particle through a fiber network" (extended Ogston model) 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 6.0.x. or 7.x, up to
256 colors or gray levels; 32 bit addressing off is the preferred
mode.
ElphoFit 2.31 is freeware and was developed at the National Institutes of
Health, Bethesda, Maryland, USA. The program is user-friendly and
provides lots of helpful comments. Additional information on 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.
Proc. Natl. Acad. Sci.USA 1970, 65: 970-977
Electrophoresis 1991 Jan;12(1):28-39
Electrophoresis 1991 Jan;12(1):46-54
Electrophoresis 1992 May;13(5):286-94
Electrophoresis 1993 Aug;14(8):720-4
Electrophoresis 1993 Mar;14(3):185-90
Anal Biochem 1987 Mar;161(2):395-411
J Chromatogr 1987 Jul 17;418:305-44 (Review)
Advances in Electrophoresis 1988, 2: 109-169 (Review)
******************************************************************
* Dietmar Tietz, Ph.D., Research Scientist *
* Biostatistics, Justus-Liebig-University *
* Ludwigstr. 27, D-35390 Giessen, Germany *
* Phone: +49-(641)-702-6015 *
* Fax: +49-(641)-702-5995 *
* Email: Dietmar.Tietz at Uni-Giessen.de *
******************************************************************
