Electrophoretic technique for separating serum proteins and improved electrophoretic gel for use therein

An electrophoretic gel of the type comprising a polysaccharide. The electrophoretic gel is characterized in that it further comprises an acid polysaccharide and salts thereof, wherein the acid moiety of the acid polysaccharide comprises at least one carboxyl group. An improved electrophoretic technique for assaying the relative distribution of proteins of the type wherein a sample to be assayed is applied to an electrophoretic gel and the electrophoretic gel is electrophoresed. The electrophoretic technique is characterized in that the above described electrophoretic gel is employed therein.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The instant invention pertains to an electrophoretic technique for 
separating serum proteins and to an electrophoretic gel for use therein. 
2. Description of the Prior Art 
Electrophoretic techniques for separating serum proteins and 
electrophoretic gels for use therein are well known to those skilled in 
the art. Cawley, Electrophoresis and Immunoelectrophoresis, Little, Brown 
and Company, Boston, Mass. (1969). In general, electrophoretic gels 
employed for separating serum proteins are of the type comprising a 
polysaccharide. A buffer having a basic pH is also commonly present in 
these electrophoretic gels. 
Typical polysaccharides employed in prior art electrophoretic gels include, 
but are not limited to, starch, cellulose acetate, agar, agarose, and 
combinations thereof. 
Typical buffers having a basic pH employed in prior art electrophoretic 
gels include, but are not limited to, the basic pH buffers which are set 
forth in Table I of Cawley, supra, pp. 331-332. 
One problem present in prior art electrophoretic techniques for separating 
serum proteins is that it takes a substantial amount of time (on the order 
of 30 minutes and longer) for the serum proteins to become fixed, i.e., 
capable of visual observation. This relatively long protein fixation 
period makes the prior art serum electrophoretic technique a time 
consuming procedure. 
Accordingly, it would be very desirable to have an electrophoretic 
technique for the separation of serum proteins wherein the protein 
fixation period is relatively short. Such an improved electrophoretic 
technique for separating serum proteins would enable the clinical 
laboratory to supply vital information to the diagnostician in a shorter 
period of time. 
SUMMARY OF THE INVENTION 
In accordance with the present invention there is provided an improved 
electrophoretic technique for separating serum proteins wherein one is 
capable of obtaining a rapid fixation of serum proteins. The 
electrophoretic technique of this invention is of the type wherein a 
sample to be assayed is applied to an electrophoretic gel and the 
electrophoretic gel is electrophoresed. The improved electrophoretic 
technique of the instant invention is characterized in that a novel 
electrophoretic gel is employed therein. The electrophoretic gel is 
characterized in that it further comprises an acid polysaccharide and the 
salts thereof, wherein the acid moiety thereof comprises at least one 
carboxyl group. It is the presence of the acid polysaccharide as well as 
the salts thereof in the electrophoretic gel which enables one to obtain 
the rapid fixation of serum proteins. 
Still other features and attendant advantages of the present invention will 
become apparent to those skilled in the art from a reading of the 
following detailed description of the preferred embodiments. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Acid polysaccharides capable of use in the instant invention include, but 
are not limited to, arabic acid, tragacanth acid, kahya acid, alginic 
acid, pectic acid, and linseed acid. The preferred acid polysaccharide is 
arabic acid. 
Salts of acid polysaccharides capable of use in the instant invention 
include, but are not limited to, the sodium, potassium, calcium, and 
magnesium salts thereof. Examples of such acid polysaccharide salts 
include, but are not limited to, arabic gum acid, tragacanth gum acid, 
khaya gum acid, alginic gum acid, pectic gum acid, and linseed gum acid. 
Polysaccharides which can preferably be employed in the electrophoretic gel 
of the instant invention are agar and agarose. The agarose can be either 
low electroendosmosis agarose, medium electroendosmosis, or high 
electroendosmosis agarose. More preferably, the polysaccharide employed in 
the electrophoretic gel of the instant invention is high electroendosmosis 
agarose. 
Preferably, the buffer employed in the instant invention has a pH of about 
7 to about 10. More preferably the buffer has a pH of about 8 to about 9. 
The electrophoretic gel of the instant invention can optionally further 
comprise a preservative agent. Typical preservative agents include, but 
are not limited to, antibiotics, halogenated organic compounds, and 
inorganic compounds. One readily available preservative agent capable of 
use herein is sodium azide. 
The electrophoretic gel of the instant invention can also optionally 
contain an alkylpolyol having 2 to 6 carbon atoms and 2 to 4 hydroxyl 
groups. Suitable alkylpolyols which can be used herein include, but are 
not limited to, ethylene glycol, propanediol, butanediol, pentanediol, and 
glycerol. Preferably, the alkylpolyol has 2 to 4 carbon atoms. 
The exact concentrations of the various constituents employed in the 
electrophoretic gel of the present invention are not critical. However, 
the electrophoretic gel of the instant invention preferably comprises from 
about 0.4 to about 1.5 percent weight/volume high electroendosmosis 
agarose; from about 0.01 to about 5 percent weight/volume arabic acid; up 
to 20 percent volume/volume ethylene glycol; up to 1 percent weight/volume 
sodium azide; and a buffer having a pH of from about 7 to about 10 and a 
molarity of about 0.001 to about 3. More preferably, the electrophoretic 
gel of the instant invention comprises from about 0.7 to about 1.2 percent 
weight/volume high electroendosmosis agarose; from about 0.5 to about 1.5 
percent weight/volume arabic acid; from about 1 to about 10 percent 
volume/volume ethylene glycol; from about 0.05 to about 0.15 percent 
weight/volume sodium azide; and a buffer having a pH of from about 8 to 
about 9 and a molarity of from about 0.05 to about 1. Optimally, the 
electrophoretic gel of the instant invention comprises about 1 percent 
weight/volume high electroendosmosis agarose; about 1 percent 
weight/volume arabic acid; about 5 percent volume/volume ethylene glycol; 
about 0.1 percent weight/volume sodium azide; and a barbital buffer having 
a pH of about 8.6 and a molarity of about 0.05. 
The electrophoretic gels of the instant invention can be prepared via any 
technique well known to those skilled in the art. See, for example, 
Cawley, supra. In general, the gel solution is prepared by mixing the 
various ingredients present therein while heating the mixture to a 
temperature of about 80.degree. to about 100.degree. C. The 
electrophoretic gel can be prepared by either standard molding or casting 
techniques. The gels can be stored at any convenient temperature, for 
example from about 2.degree. to about 40.degree. C., preferably from about 
18.degree. to about 26.degree. C. It is preferred to store the 
electrophoretic gels in sealed, plastic trays until ready for use. 
Samples can be applied to the electrophoretic gels of the instant invention 
via any technique used in the prior art, e.g., via a microliter syringe. 
The electrophoretic gels can be electrophoresed at 100 volts for 20 
minutes. If desired, the gels can be fixed in an alcohol:acetic acid 
mixture such as 60 percent reagent alcohol, 30 percent deionized water, 
and 10 percent glacial acetic acid. In addition, the gels can optionally 
be dried at about 80.degree. to about 90.degree. C.

The following examples are provided for the purpose of further illustration 
only and are not intended to be limitations on the disclosed invention. 
EXAMPLE 1 
The two electrophoretic gel formulations set forth in Table I were each 
employed in the following protocol in order to demonstrate the improved 
electrophoretic technique of the instant invention for separating serum 
proteins and the improved electrophoretic gel for use therein. The sole 
difference between the two electrophoretic gels employed in this 
comparative experiment was that the electrophoretic gel within the scope 
of this invention contained arabic acid, i.e., a specific acid 
polysaccharide, whereas the electrophoretic gel outside of the scope of 
this invention was devoid of any acid polysaccharide. 
PROTOCOL 
Electrophoretic Procedure 
1.0 A serum sample was applied to the surface of the gel via a template 
method. 
2.0 The gel was electrophoresed in barbital buffer, pH 8.6. 
3.0 The gel was placed in a fixative solution comprising deionized water, 
ethyl alcohol, and glacial acetic acid (3:6:1) until precipitated and 
observed. 
The results obtained from the above protocol for each gel formulation of 
Table I are also set forth in Table I. 
TABLE I 
______________________________________ 
Electrophoretic 
Electrophoretic Gel 
Gel 
Within Scope of 
Outside Scope of 
Ingredients Instant Invention 
Instant Invention 
______________________________________ 
1% wt/v agarose 
(high electroendosmosis) 
X X 
1% wt/v arabic acid 
X -- 
0.1% wt/v sodium azide 
X X 
5% v/v ethylene glycol 
X X 
Barbital buffer, pH 8.6 
.+-. 0.1 at 20-25.degree. C.; 
ionic strength 0.05 
X X 
Serum Protein Fixation 
Time (t), Minutes 
t&lt;3 t&gt;30 
______________________________________ 
As the serum protein fixation times of Table I demonstrate, an 
electrophoretic technique for separating serum proteins employing the 
improved electrophoretic gel of the instant invention is capable of fixing 
serum proteins in less than 3 minutes. In contrast, a similar technique 
differing solely in that the electrophoretic gel employed therein was 
devoid of any acid polysaccharide (wherein the acid moiety thereof 
comprises at least one carboxyl group) or the salts thereof was incapable 
of fixing the serum protein even after a 30 minute period. 
Based on this disclosure, many other modifications and ramifications will 
naturally suggest themselves to those skilled in the art. These are 
intended to be comprehended as within the scope of this invention.