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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention broadly relates to a method and apparatus for
conducting gel electrophoresis. The invention specifically relates to a
method and apparatus for resolving a complex mixture of components into a
fixed two-dimensional array of its constituents in a gel matrix. In its
preferred form, the invention pertains to a method and apparatus for the
separation of complex mixtures of bio-organic molecules such as proteins
and nucleic acids on the basis of two independent physical
characteristics.
2. Description of the Prior Art
At present, in order to obtain very high resolution in the separation of
bio-organic molecules one generally must employ the technique of
two-dimensional gel electrophoresis first described by P. H. O'Farrel,
(1975) Journal of Biological Chemistry, 250:4007-4021. By utilizing this
technique, exceedingly complex mixtures of bio-organic molecules,
containing for example over 1,000 different proteins, are separated and
analyzed at one time.
According to this delicate, labor-intensive and time-consuming procedure,
components of a protein mixture are separated in a two-step procedure
which first involves a partition of proteins as a function of net surface
charge by isoelectric focusing (IEF) in one dimension, followed by
separation as a function of size (by molecular sieving in a second
dimension.
This separation process is carried out in a jelly-like material called a
polyacrylamide gel. This gel is cast by polymerization of a mixture of
acrylamide monomer and an appropriate cross-linking agent, such as
N,N,N',N'-tetramethylethylene-diamine (TEMED), in suitably sized glass
tubes or between rectangular glass plates to form rods or thin sheets,
respectively.
Isoelectric focusing (IEF), the first separation step, relies on the fact
that bio-organic molecules such as proteins and peptides, are
three-dimensional objects with ionizable surface groups (e.g., carboxyl,
amino, imidazole, guanidinium, etc.). These ionizable groups are
amphoteric in nature; i.e., below a certain pH, such groups are positively
and above a certain pH, they are negatively charged. At a particular pH
value, called the isoelectric point (pI), the number of positively charged
surface groups equals the number of negatively charged surface groups on
the molecule. Consequently, the molecule will have a net charge of zero.
IEF involves the electrophoretic migration of a molecule in a pH gradient
until it reaches the pH corresponding to its isoelectric point. As the net
charge of the molecule is zero at that point, it becomes immobilized and
remains "focused" at its respective pI.
Stable pH gradients are generated within polyacrylamide gels by inclusion
of specialized buffer constituents called ampholytes. Ampholytes consist
of a mixture of low molecular weight amphoteric compounds with isoelectric
points covering a defined range of pH values. Application of a
constant-voltage, DC electric field to an ampholytebuffered gel causes the
ampholytes to focus according to their pIs, thereby establishing a pH
gradient with sufficient buffering capacity and conductance for subsequent
focusing of amphoteric macromolecules.
After performing the first dimension IEF separation in a gel, cast in a
glass tube, the technician performing the separation then must carefully
remove the gel rod, which contains the focused proteins, from the glass
tubing.
Typically, each gel rod then is treated with a second dimension
electrophoresis buffer solution. This aqueous buffer solution contains a
detergent such as sodium dodecyl sulfate (SDS), which binds to proteins in
the gel providing these macromolecules with a net negative charge.
Afterwards, each gel sample is placed on top of and cemented to one edge
of a second dimension polyacrylamide gel slab.
The assembled composite consisting of the treated first dimension gel
overlaying and secured to the second dimension gel slab then is placed
within an electric field between appropriate electrically conductive
buffer solutions and separation in the second dimension may proceed on the
basis of the proteins' differing molecular sizes via the process of
molecular sieving.
Depending upon the particular apparatus design and gel and buffer
chemistries, the technique of two-dimensional gel electrophoresis requires
anywhere from about 17 hours to more typically about 30 hours to perform
each separation. Moreover, not only is this separation procedure quite
labor-intensive and time-consuming, but as those skilled in this
technology readily appreciate the procedure also demands very high
expertise and exacting laboratory techniques on behalf of each operator to
ensure reproducibility of results. Unavoidably, the technique also places
the fragile polyacrylamide gel containing the separated components
generated in the first dimension at considerable risk during the transfer
process preparatory to the second dimension separation.
As a consequence of these substantial drawbacks, two-dimensional gel
electrophoresis has not been used for many applications where the
procedure is ideally suited, such as for example clinical diagnostic
screening and other larger scale analytical applications.
Another separation technique useful for separating bio-organic molecules
which continues to receive considerable attention from both the basic
researcher and clinical diagnostician is high performance liquid
chromatography (HPLC). Although incapable of achieving the same degree of
resolution possible with two-dimension gel electrophoresis, this procedure
nevertheless provides a very high resolution of the components in a
complex mixture. In fact, recent advances in this separation technology
have virtually eliminated the gap between this technique and
one-dimensional electrophoresis techniques. More importantly, when using
this technology the separation of complex mixtures of bio-organic
molecules is achieved in only a fraction of the time necessary to carry
out the process of gel electrophoresis.
According to this technique, a mobile phase or eluate, into which the
sample to be analyzed has been injected, is forced through a bed of
microparticulate chromatographic packing material at a relatively high
linear velocity. Velocities on the order of 0.1 millimeter/second or
greater are typical. As recognized by those skilled in this art,
separation of components in the sample depends in large part upon eluate
chemistry and the nature of the packing material used. Proteins and other
bio-organic molecules are separated in this procedure, inter alia, on the
basis of size (gel permeation chromatography), ionic properties
(ion-exchange), absorptive characteristics (absorption chromatography) and
hydrophobicity (reversed phase chromatography).
Analysis of the separated components typically is accomplished using
in-line detectors measuring such component properties as absorbance and
refractive index. It also is known to couple the detector and the
chromotagraphic column through a post-column reactor wherein separated
components of the sample are converted, by reaction with appropriate
reagents, into fluorescent or color derivatives. The so-altered species
then can be identified using an appropriate detector.
One object of the present invention is to provide a method and apparatus
for automating gel slab electrophoresis.
Another object of the present invention is to provide a method and
apparatus suitable for sequentially resolving a complex mixture of
components and particularly a mixture of bio-organic molecules, preferably
according to two independent physical characteristics.
A further object of this invention is to provide a method and apparatus for
separating a complex mixture of components, particularly a mixture of
bio-organic molecules, which method and apparatus exhibit advantages of
both high performance liquid chromatography and two-dimensional gel
electrophoresis techniques.
Yet a further object of this invention is to provide method and apparatus
which automate the sequential separation of a complex mixture of
components, particularly a mixture of bio-organic molecules, that has
heretofore been accomplished using the well-known manual procedure of
two-dimensional gel electrophoresis, without losing the resolving power
inherent in such well-known technique.
These and other objects of this invention will become apparent from a
consideration of the specification and appended claims.
SUMMARY OF THE INVENTION
In a first aspect, the present invention broadly relates to a method for
loading the components of a sample mixture into an electrophoresis gel
slab which comprises:
(a) forming said sample into a flowing stream of fluid,
(b) treating said flowing stream of fluid, as required, so that the
components in said flowing stream exhibit a substantially uniform surface
charge density; and
(c) aligning the flow of said stream containing said charged components
incident to an inlet face of a gel slab by moving a source of said flow
relative to the inlet face of said gel slab, said gel slab being
positioned in an electric field having a sufficient field strength to
force said charged components from said stream into said gel slab
substantially simultaneously with exposure of said charged components to
said electric field.
In another aspect, the present invention comprises a method for separating
the components of a sample mixture comprising:
(a) treating said sample so as to generate a treated fluid sample having
its components longitudinally separated on the basis of a common physical
characteristic of said components;
(b) recovering said treated fluid sample as a flowing stream of fluid;
(c) treating said flowing stream stream, as required, so that the
longitudinally separated components in said flowing stream exhibit a
substantially uniform surface charge density, said treatment being
accomplished with substantially no loss in the separation obtained in step
(a);
(d) aligning the flow of said stream containing said longitudinally
separated and charged components incident to an inlet face of a gel slab
by moving a source of said flow relative to the inlet face of said gel
slab, said gel slab being positioned in an electric field having a
sufficient field strength to force said charged components from said
stream into said gel slab with substantially no loss of the separation
obtained in step (a) substantially simultaneously with exposure of said
longitudinally separated and charged components to said electric field;
and
(e) resolving said components in said gel slab on the basis of a common
physical characteristic of said components.
Preferably, separation of the components in the sample in steps (a) and (e)
is on the basis of two independent physical characteristics.
In a third aspect, the present invention comprises an apparatus for loading
the components of a sample mixture into an electrophoresis gel slab
comprising:
(a) first conduit means for transfering said sample mixture as a flowing
stream of fluid;
(b) associated pump and mixer means for treating said flowing stream
transferred in said first conduit means with a reagent stream so that the
components in said flowing stream exhibit a substantially uniform surface
charge density;
(c) a gel slab suitable for gel electrophoresis;
(d) second conduit means for conducting said flowing stream of said charged
components to a gel tracking carriage assembly;
said gel tracking carriage assembly positioned over an inlet of said gel
slab and adapted for movement along the inlet face of said gel slab,
said gel tracking carriage assembly including an outlet nozzle for aligning
said flowing stream containing said charged components incident to said
inlet face, the end of said nozzle being closely spaced from and oriented
substantially parallel to the inlet face of said gel slab and
(f) means for generating an electric field in said gel slab having a
sufficient field strength to force said charged components from said
flowing stream into said gel slab substantially simultaneously with
exposure of said charged components to said electric field.
In a fourth aspect, the present invention also comprises an apparatus for
separating the components of a sample mixture comprising:
(a) column means for longitudinally separating the components in a fluid
sample on the basis of a common physical characteristic of said
components;
(b) first conduit means for recovering a treated fluid sample from said
column as a flowing stream of fluid;
(c) associated pump and mixer means for treating said flowing stream
recovered in said first conduit means with a reagent stream so that the
separated components in said flowing stream exhibit a substantially
uniform surface charge density, said associated pump and mixer means
accomplishing said treatment with substantially no loss in the separation
of said components;
(d) a gel slab suitable for gel electrophoresis;
(e) second conduit means for conducting said flowing stream of said
longitudinally separated and charged components to a gel tracking carriage
assembly;
said gel tracking carriage assembly positioned over an inlet face of said
gel slab and adapted for movement along the inlet face of said gel slab
said gel tracking carriage assembly including an outlet nozzle for aligning
said flowing stream containing said longitudinally separated and charged
components incident to said inlet face, the end of said nozzle being
closely spaced from and oriented substantially parallel to the inlet face
of said gel slab, and
(f) means for generating an electric field in said gel slab having a
sufficient field strength to force said charged components from said
flowing stream into said gel slab with substantially no loss in the
longitudinal separation of said components substantially simultaneously
with exposure of said longitudinally separated and charged components to
the electric field.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic illustration of apparatus useful for carrying out the
preferred method of this invention.
FIGS. 2-4 illustrate the outlet nozzle of the gel tracking carriage
assembly in relation to the inlet face of the electrophoresis gel slab.
FIG. 4, in particular, is an enlarged view of the outlet nozzle and inlet
face of the gel slab.
FIG. 5 schematically illustrates another apparatus useful for practicing a
preferred embodiment of the method of the present invention.
DETAILED DESCRIPTION
The present invention pertains to a method and apparatus suitable for
separating complex mixtures of components such as bio-organic molecules,
particularly including proteins or nucleic acids, preferably on the basis
of two independent physical characteristics of said components. FIG. 1
schematically illustrates one embodiment for carrying out the method of
this invention.
In this particular embodiment, a high performance liquid chromatography
column 60 is used to effect the first stage separation. As shown in FIG.
1, an isocratic elution stream obtained from reservoir 10 is pumped
through a sample injection valve 30 using a high pressure, low impulse,
constant flow pump 20 such as the Beckman Instruments Model 114M Solvent
Delivery System. The sample containing the mixture of components to be
separated is injected into the sample injection valve 30 through conduit
40 and is carried by the elution stream into the HPLC column 60 through
conduit 50. As will be appreciated by those skilled in this technology, an
elution stream composition must be selected which does not interfere with
the second stage separation (electrophoresis). Appropriate compositions
can be obtained using routine experimentation.
The actual design of the HPLC column and the particular microparticulate
packing employed forms no part of the present invention. Any of the wide
variety of commercially available hardware could be used in concert with
microparticulate column packing materials such as the Pharmacia Fine
Chemicals MonoBead (TM) line of chromatographic adsorbents specifically
designed for peptide and protein chemistry. In HPLC column 60, different
components in the sample are separated longitudinally from one another on
the basis of a common physical characteristic or characteristics such as
for example size, ionic or absorptive characteristics, hydrophobicity or a
combination thereof. As the elution stream continues to be pumped from
reservoir 10 different components in the originally injected sample move
through the column bed at different velocities as a consequence of such
different property(ies) thereby causing these components to separate. The
components in the original sample appear as longitudinally spaced
chromatographic waves or bands in the fluid stream or treated fluid sample
(generally a liquid) flowing out of the HPLC column in conduit 70. The
treated fluid sample containing the longitudinally separated components is
recovered as a continuously flowing stream of fluid in conduit 70.
In the second stage separation, the longitudinally separated components
then are resolved in a direction orthogonal to the separation obtained by
the first stage separation. As shown in FIG. 1 embodiment, this separation
is accomplished by gel electrophoresis, preferably using a controlled
porosity polyacrylamide gel material. In the broad practice of this
invention, the second stage gel electrophoresis may be run using any gel
medium conventionally employed, such as polycrylamide. Preferably, as
shown in FIGS. 2 and 3, the gel slab consists of two gel phases a stacking
gel (capture medium) and a separating gel, such composites are well-known
to those skilled in this technology and require no further description.
Before entering the second stage separation the longitudinally separated
components obtained in the first dimension separation are treated, as
required, so that the components exhibit a substantially uniform net
surface charge density. For example, treatment generally is required if a
protein-containing sample is being separated. Treatment generally would
not be required if a mixture of nucleic acids was being separated.
Proteins are relatively large bio-organic molecules composed of large
numbers of amino acids residues. Proteins assume exceedingly complex
conformations, and generally do not exhibit a uniform surface charge
density in aqueous medium. Consequently, in order to effectuate further
separation of the proteins in the second stage gel matrix it is necessary
to treat the proteins so as to produce a uniform charge environment.
A uniform surface charge density can be imposed in a satisfactory manner on
the protein components by using any of a variety of protein denaturing
solutions well-known in the art, e.g., the detergent sodium dodecyl
sulfate (SDS), and the reducing agent 2-mecaptoethanol. In the FIG. 1
embodiment, this treatment is accomplished by pumping a denaturing
solution from reservoir 80 using constant flow, low pulse output pump 90
into mixer 100 where the denaturing solution is blended with the
continuously flowing stream of the treated fluid sample containing the
longitudinally separated components. The denaturing solution stream is
blended into the treated fluid sample in a manner which does not
substantially degrade the resolution obtained in the first dimension HPLC
separation. The detergent component of the denaturing solution (e.g., SDS)
coats the protein components in the treated sample so as to yield a uniform
charge per unit mass environment. The treated fluid sample then can be
passed through a heat exchanger 110, if necessary, to activate the
detergentprotein interaction. The sample processing steps carried out by
means of the mixer 100 and heat exchanger 110 can be achieved by a wide
variety of commercially available post-column reactors such as the Beckman
Instruments Model 230 system.
The continuously flowing stream now containing longitudinally separated and
charged components then is passed to the second stage gel 120. The
separation in the second stage is accomplished on the basis of differing
sizes of the longitudinally separated components of the fluid sample. As
recognized by one skilled in this technology, the resolution obtained in
the second stage separation requires use of a gel matrix material having a
pore size controlled to approximate the size of the components being
separated. In this way, molecular sieving occurs and separation in the gel
occurs on the basis of molecular size. Preferably, the first stage
separation was accomplished on the basis of an independent physical
characteristic.
Referring now to FIGS. 2 through 4, the procedure for transfering the
longitudinally separated and charged components in the continuously
flowing stream to the second stage gel slab is illustrated. As shown in
FIG. 2, a gel slab 200 is interposed in an electric field (D.C.) having
opposite edges in contact with appropriate electrically conductive buffer
solutions 201 and 202. The continuously flowing stream preferably having a
density equal to or greater than the density of the upper buffer solution,
is aligned incident to the inlet face of the gel slab by flowing the
treated stream through an appropriately configured outlet nozzle 205 of a
gel tracking carriage assembly (not shown) and simultaneously moving the
gel tracking carriage assembly along the inlet face of the gel slab. As
shown, the inlet face of the gel slab comprises a stacking gel or capture
medium 203, which overlays the separating gel 204. The outlet 206 of the
nozzle 205 is oriented substantially parallel to and positioned in close
proximity to the inlet face of the gel slab (e.g., about 0.1 to 0.5 mm
above the face of the gel slab).
The gel tracking carriage assembly traverses the inlet face of the gel slab
at a rate coordinated with the total time required for longitudinal
separation of the sample components in the first stage separation. Means
for moving the gel tracking carriage assembly across the face of the gel
slab in coordination with the total time required for the first stage
separation, including the necessary motive means and carriage assembly,
will be apparent to those skilled in the art. In this way the
longitudinally separated components are aligned incident to the entire
length of the inlet face of the gel slab. By maintaining an electric field
across the gel slab of a sufficient field strength, the longitudinally
separated and charged components in the fluid stream are forced directly
from the fluid sample stream into the gel slab substantially
simultaneously with the exit of said stream from the outlet nozzle and
exposure of the separated and charged components to the electric field
such that there is substantially no loss in the separation (resolution)
obtained inthe first stage. This process is schematically illustrated in
FIG. 4.
The direct current power requirements for loading the separated components
of the treated fluid sample into the gel slab typically will be on the
order of about 25 watts. The same (or different) requirements then can be
used to perform the second stage electrophoresis separation. An
appropriate power for conducting specific separations can be determined
using routine experimentation. Apparatus available in the prior art for
imposing an electrical field across a gel slab and for cooling the gel
slab, if required, during operation, such as the Pharmacia Model GE-2/4
Vertical Slab Gel Electrophoresis unit can be used in practicing the
present invention.
By this arrangement, the resolution achieved in the first stage separation
is preserved substantially completely upon the transfer of the sample to
the second stage separation.
As shown more clearly in FIG. 3, the inlet face of the separation gel
preferably is provided with a different gel chemistry (pore structure and
electrolyte buffer composition), to maximize the rate that the charged
components of the treated fluid sample are driven into the second stage
gel. Such gel electrolyte systems, described as multiphasic zone
electrophoresis systems (cf. T. M. Jovin, Biochemistry 12: 871-898), are
known in the prior art. One such gel chemistry system is that of U. K.
Laemmli (Nature 227: 680-685 [1970]). Gels of this type are preferred for
use in the present invention.
After loading the charged components of the fluid sample into the inlet
face of the second stage gel slab, the gel now is ready for initiation of
the second stage electrophoretic separation. This separation can be
conducted immediately after loading the sample components into the gel or,
if desired, the gel can be stored under appropriate conditions and the
separation conducted at a latter time together with numerous other gels
prepared in a similar fashion. The option of storing numerous gels for
later separation en masse is an important advantage of the present
invention.
Once the second stage electrophoteric separation is complete, visualization
or recovery of the separated components in the gel slab can be accomplished
for example using standard techniques of colorimetry, radiology and
extraction.
In the broad practice of this invention, any procedure for separating
components in a fluid, e.g., liquid, sample in a longitudinal fashion so
as to produce a continuously flowing stream of fluid can be used for the
first stage separation. As used throughout the specification and claims,
the term "longitudinal" broadly refers to the process of separating a
mixture of components in a fluid, e.g., liquid, sample into a treated
sample that contains a one-dimensional spaced array of said components.
The term "longitudinal" is not used to characterize the uniformity of the
spacing between individual separated components but rather is used in a
broader sense to emphasize the nature of the separation, that is that the
separation is accomplished in a single dimension. While any of the wide
variety of HPLC techniques preferably are used for the first stage
separation, the present invention also contemplates using other available
techniques for longitudinally separating components in a fluid stream,
such as any of the known electrophoresis techniques. The necessity of
generating a fluid stream, of course, excludes gel electrophoresis for the
first stage separation however.
Referring next to FIG. 5, a preferred embodiment for carrying out the
method of the present invention is schematicaly illustrated. The major
elements of this preferred arrangement include an array of HPLC columns
for conducting the first stage separation; the equipment needed to prepare
the output of the first stage separation for the second stage gel
electrophoresis separation including the reagent supply and the necessary
pumps, mixers and heat exhange equipment, and the second stage
electrophoresis gel arrangement including the gel itself, the gel tracking
carriage assembly for loading the output of the first stage separation on
the gel and the means for generating an electric field (D.C.).
As in the FIG. 1 embodiment, the preferred procedure for conducting the
first stage separation is high performance liquid chromatography. In the
FIG. 5 embodiment, however, this separation is accomplished by using
gradient elution. As shown, the elution stream for the first stage
separation is prepared by blending a portion of the elution solvent in
reservoir 301 with a portion of the elution solvent in reservoir 302 both
fed to mixer 305 using pumps 303 and 304, respectively. By separately
controlling the pumping rates of pumps 303 and 304 any of a wide range of
continuous and discontinuous elution gradients can be prepared. As is
known in the prior art, the composition of elution solvents in reservoir
301 and 302 are constrained by the electrolyte combinations used for
carrying out the second stage electrophoresis operation as specified by
the Multiphasic Buffer Systems Output developed by T. M. Jovin, M. L.
Dante and A. Chambrach, available as documents PB No. 196085 to 196092 and
203016 from the National Technical Information Service, Springfield,
Virginia.
the mixed eluate stream so-prepared then is passed through a pre-column 306
for removing any solvent impurities. The elution stream is introduced into
a manifold 307 which partitions the elution stream prior to its entry into
the multiple sample injection valves 308 for flow into the parallel array
of HPLC columns 309. In this arrangement, multiple samples, e.g., blood
serum samples from a large number of patients, are separately injected
into each partitioned elution stream and each sample then is delivered to
its respective HPLC column.
As in the FIG. 1 embodiment, as each elution stream flows through its
respective HPLC column, different components in the injected sample tend
to move at different rates through the chromotographic bed as a
consequence of their different properties, causing the components to be
longitudinally separated. The fluid stream containing the longitudinally
separated components then flows from each HPLC column 309 through conduits
310 to an associated mixing device 311 where a reagent stream, e.g., SDS,
obtained from reservoir 312, necessary to yield a uniform surface charge
density on the separated components in the fluid stream is blended with
the longitudinally separated sample. The reagent stream is fed to the
respective mixers 311 from reservoir 312 using pump 313. Each continuously
flowing stream then passes through heat exchanger 314 to activate the
reagent and complete the binding of SDS to the separated components in the
treated fluid.
Each treated fluid stream then is passed to the gel tracking assembly 315
and the treated components of each fluid stream are aligned incident to an
inlet face of a gel slab, indicated schematically as element 316, as
described in connection with the FIG. 1 embodiment. Each gel is exposed to
a D.C. field generated using power supply 317. Using this arrangement,
multiple samples can be separated on the basis of two independent physical
characteristics using gel electrophoresis in only a fraction of the time
required in the prior art.
In this preferred embodiment a micro-processor typically will provide a
complete sequence of operations needed to process samples through the
first and second stage separations, including the generation of the
elution gradient for the first stage separation, sample injection,
treatment of the first stage output with reagent and controlling the gel
tracking carriage assembly and power supply for transfering the treated
first stage output into the second stage gel slab by electromotive force.
In the practice of the present invention, a wide variation in the
procedures for conducting the various steps of the inventive method are
possible. For example, in addition to using high performance liquid
chromatography for the first stage separation, the present invention also
contemplates the use of electrophoresis such as isoelectric focussing
using pH gradient and density gradient techniques.
While the present invention has been described with respect to preferred
embodiments and particularly to the separation of the components in a
complex mixture, it should be understood that various changes may be made
without departing from the spirit and scope of the invention as
particularly claimed below. For example, another aspect of this invention
relates to the method and apparatus for loading the components of a sample
into an electrophoresis gel slab.
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