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New simple technique for the multifractionation of serum proteins (18–21 fractions) on Cellogel RS cellulose acetate strips
CLINICA
475
CHIMICA ACTA
NEW SIMPLE TECHNIQUE PROTEINS
(18-21
FOR THE MULTIFRACTIONATION
FRACTIONS)
OF SERUM
ON CELLOGEL RS CELLULOSE
ACETATE
STRIPS
G. B. DEL
Laboratory (Received
CAMP0
ofElectrophoretic
and Chromatographic
Technology, Chemetron, Milan
(Italy)
April zz, 1968)
SUMMARY
A new supporting medium: Cellogel RS, a special cellulose acetate, is proposed for the separation of serum proteins into 18-21 fractions by conventional electrophoresis. The method is described in detail and some advantages and differences compared with previous systems using starch gel or polyacrylamide gel are described.
INTRODUCTION
The multifractionation of serum proteins by electrophoresis on cellulose acetate did not seem feasible until now. But Cellogel RS strips (a special kind of gelatinized cellulose acetate) easily allows the separation of 18-21 fractions by conventional electrophoresis on cellulose acetate. The method is highly sensitive: using only 2.5 ~1 of serum, one can detect fractions representing less than 0.5 y0 of total proteins. The Cellogel strip is not rendered transparent but treated in a special way by which also small fractions become optically intensified. The electrophoretic run is 11-12 cm long, and it is carried out in I 4 or 2 hours. The staining and destaining process is the same as that used for cellulose acetate and needs only a few minutes. Practical advantages of this new technique compared with the two techniques known so far (multifractionation of serum proteins on starch gel according to Smithies (ref. I) and multifractionation on polyacrylamide gel according to Raymond and Weintraub2 or Ornstein and Davis3) are the following: (I) it does not require any unusual procedure, being the commonly used, wellknown electrophoresis on cellulose acetate. (2) The medium does not influence the fractionation path with a molecular sieving effect. The lipoprotein fraction does not undergo the adsorbing effect which is typical of highly hydrophilic gels. These fractions appear in the electrophoretic positions indicated by Colfs and Verheyden4. Indeed, Cellogel is a medium hydrophilic Cl&. Chim. Acta, 22 (1968) 475-479
DEL CAMP0
476
Fig. I. Conventional electrophoretic pattern of protein molecules on Cellogel RS, according to electric charge. Each zone is further subdivided, e.g. the albumin zone shows 3 fractions: prealbuminic halo (lipidic), albumin and postalbumin (lipoprotein).
as well as lipophilic, where both lipidic and non-lipidic proteins have a good migration. (3) The intensity and position of fractions agree with immunoelectrophoresis on cellulose acetate, when the latter is done with total antiserumS~6.Complexes Hp-Hb migrate in the u2position, demonstrating the absence of the molecular sieving effect. (4) At the end of the electrophoretic run one can apply special staining procedures on the strip. This is very difficult and sometimes impossible on starch gel or polyacrylamide gel. (5) Dry Cellogel RS strips can be stored indefinitely for documentation purposes. MATERIALS
* * * * *
Standard power supply 0-400 V, o-50 mA. Chemetron electrophoretic tank. Serum double wire applicator (2.5 ~1). Cellogel RS strips 3 x 23 cm (with wedge-shaped extremity). VTter buffer pH 8.9 (tris-barbital buffer 0.036 M). Carton filter rectangles 3,5 X 22 cm.
* Supplied all over the world by the distributors C&n. Chim. Acta,
22
(1968) 475-479
of the firm Chemetron,
Via G. Modena 24, Milan.
FRACTIONATION
477
OF SERUM PROTEIh’S Oh’ CELLoGEL
&rips.
* Ponceau S stain (0.5 g in IOO ml 5 o/0trichloroacetic acid). 5 y0 Acetic acid for destaining. 40 y0 Formaldehyde for stronger fixation of stained fractions. 7 O/O Glycerol in water. METHOD I. Submerge 4 Cellogel strips in 250 ml of buffer for 15 min. 2. Pour in the tank 750 ml of buffer. 3. Eliminate the excess of buffer from the strips between two sheets of filter paper and place the strips on the bridge. Apply at the hanging edges the carton filter wicks (wetted in the buffer). 4. Apply the serum with sample applicator at the cathodic end. 5. Run at 400 V for z h (or at 450 V for go min or at 300 V for 2 h and 40 min). 6. On completion of the run, stain the strip for 5 min. Destain with 3-4 baths of decolorant solution. 7. Fix in I00 ml of formalin for 2 min. 8. Submerge in 2 baths of 7 y0 glycerol (zoo ml) for 3 min. g. Lay the strips on a glass plate. Eliminate the excess of glycerol solution with a filter paper and warm at go’ (5-6 min) until the strips become completely white.
* G. T. Gurr, x2/16 Carlisle Road, Colindale N.W., London (U.K.). C&in.Chim. Acfa, 22 (1968) 475-479
DEL CAMP0
Fig. 3. Separation of 4 sera on a single Cellogel RS strip. Conditions: 400 V for I h in VTter buffer, pH 8.9 (tris-barbital buffer 0.035 M). The strip is placed on a bridge of 14 cm. The electrophoretic pathway is g cm long. Samples are applied with the Chemetron microapplicator (0.5 ,ul/5 mm). IO. Remove
the strips while they are still hot and examine
them against
the
light of a 60-100 W bulb at a few cm distance. EXPERIMENTAL
RESULTS
All separated fractions are detected only at the end of the bleaching process. This treatment produces a swelling of the strip and a photomultiplication of the coloration. The pattern in Fig. I is typical of a normal serum. The Cellogel RS pattern is very different from a starch gel or polyacrylamide gel pattern. This is due to the fact that the Cellogel RS pattern does not show an enrichment in the y zone produced by the molecular sieving effect and the hydrophilic action which is typical of the other gels. The bands on Cellogel RS are very sharp in spite of the long migration (II--12 cm), because the porosity of this product decreases from one surface (penetrable by proteins) to the other (impenetrable). In this way diffusion is hindered. Procedures apt to detect lipo-4 and glycoproteins7 are applied if the electrophoretic run is carried out with a two-fold sample application (5 ~1). REFERENCES I 0. SMITHIES, Biochem. J., 61 (1955) 629. 2 S. RAYMOND AND L. WEINTRAUB, Science, 130 (1959) 711. Clin. Chim. Ada,
22 (1968) 475-479
FRACTIO~ATIO~
OF SERUM PROTEINS
ON CELLOGEL
479
3 L. ORNSTEIN AND B. DAVIS,Anti. N. Y. Acad.Sci., 121 (1964) 321,404. 4 B. COLFS AND J.VERHEYDEN,CE~~.C~W. Acta, IZ (1967)470. 5 W. MULLER BEISSENHIRTZAND H. KELLER,C&Z. Chim. Ada, 13 (1966) 95. 6 B. LOMANTO, C. VERGANI AND L. MASCARETTI,Atti Accad. Med. Lombarda, 20 (1965); Clin. Chim. Acta, 15 (1967) 169. 7 M.PAGET AND P.COUSTENOBLE,Ann.Biol.Clin. (Paris), 23 (1~65) Izog.
Clin.Chim. Acta, 22 (1~68)475-479
475
CHIMICA ACTA
NEW SIMPLE TECHNIQUE PROTEINS
(18-21
FOR THE MULTIFRACTIONATION
FRACTIONS)
OF SERUM
ON CELLOGEL RS CELLULOSE
ACETATE
STRIPS
G. B. DEL
Laboratory (Received
CAMP0
ofElectrophoretic
and Chromatographic
Technology, Chemetron, Milan
(Italy)
April zz, 1968)
SUMMARY
A new supporting medium: Cellogel RS, a special cellulose acetate, is proposed for the separation of serum proteins into 18-21 fractions by conventional electrophoresis. The method is described in detail and some advantages and differences compared with previous systems using starch gel or polyacrylamide gel are described.
INTRODUCTION
The multifractionation of serum proteins by electrophoresis on cellulose acetate did not seem feasible until now. But Cellogel RS strips (a special kind of gelatinized cellulose acetate) easily allows the separation of 18-21 fractions by conventional electrophoresis on cellulose acetate. The method is highly sensitive: using only 2.5 ~1 of serum, one can detect fractions representing less than 0.5 y0 of total proteins. The Cellogel strip is not rendered transparent but treated in a special way by which also small fractions become optically intensified. The electrophoretic run is 11-12 cm long, and it is carried out in I 4 or 2 hours. The staining and destaining process is the same as that used for cellulose acetate and needs only a few minutes. Practical advantages of this new technique compared with the two techniques known so far (multifractionation of serum proteins on starch gel according to Smithies (ref. I) and multifractionation on polyacrylamide gel according to Raymond and Weintraub2 or Ornstein and Davis3) are the following: (I) it does not require any unusual procedure, being the commonly used, wellknown electrophoresis on cellulose acetate. (2) The medium does not influence the fractionation path with a molecular sieving effect. The lipoprotein fraction does not undergo the adsorbing effect which is typical of highly hydrophilic gels. These fractions appear in the electrophoretic positions indicated by Colfs and Verheyden4. Indeed, Cellogel is a medium hydrophilic Cl&. Chim. Acta, 22 (1968) 475-479
DEL CAMP0
476
Fig. I. Conventional electrophoretic pattern of protein molecules on Cellogel RS, according to electric charge. Each zone is further subdivided, e.g. the albumin zone shows 3 fractions: prealbuminic halo (lipidic), albumin and postalbumin (lipoprotein).
as well as lipophilic, where both lipidic and non-lipidic proteins have a good migration. (3) The intensity and position of fractions agree with immunoelectrophoresis on cellulose acetate, when the latter is done with total antiserumS~6.Complexes Hp-Hb migrate in the u2position, demonstrating the absence of the molecular sieving effect. (4) At the end of the electrophoretic run one can apply special staining procedures on the strip. This is very difficult and sometimes impossible on starch gel or polyacrylamide gel. (5) Dry Cellogel RS strips can be stored indefinitely for documentation purposes. MATERIALS
* * * * *
Standard power supply 0-400 V, o-50 mA. Chemetron electrophoretic tank. Serum double wire applicator (2.5 ~1). Cellogel RS strips 3 x 23 cm (with wedge-shaped extremity). VTter buffer pH 8.9 (tris-barbital buffer 0.036 M). Carton filter rectangles 3,5 X 22 cm.
* Supplied all over the world by the distributors C&n. Chim. Acta,
22
(1968) 475-479
of the firm Chemetron,
Via G. Modena 24, Milan.
FRACTIONATION
477
OF SERUM PROTEIh’S Oh’ CELLoGEL
&rips.
* Ponceau S stain (0.5 g in IOO ml 5 o/0trichloroacetic acid). 5 y0 Acetic acid for destaining. 40 y0 Formaldehyde for stronger fixation of stained fractions. 7 O/O Glycerol in water. METHOD I. Submerge 4 Cellogel strips in 250 ml of buffer for 15 min. 2. Pour in the tank 750 ml of buffer. 3. Eliminate the excess of buffer from the strips between two sheets of filter paper and place the strips on the bridge. Apply at the hanging edges the carton filter wicks (wetted in the buffer). 4. Apply the serum with sample applicator at the cathodic end. 5. Run at 400 V for z h (or at 450 V for go min or at 300 V for 2 h and 40 min). 6. On completion of the run, stain the strip for 5 min. Destain with 3-4 baths of decolorant solution. 7. Fix in I00 ml of formalin for 2 min. 8. Submerge in 2 baths of 7 y0 glycerol (zoo ml) for 3 min. g. Lay the strips on a glass plate. Eliminate the excess of glycerol solution with a filter paper and warm at go’ (5-6 min) until the strips become completely white.
* G. T. Gurr, x2/16 Carlisle Road, Colindale N.W., London (U.K.). C&in.Chim. Acfa, 22 (1968) 475-479
DEL CAMP0
Fig. 3. Separation of 4 sera on a single Cellogel RS strip. Conditions: 400 V for I h in VTter buffer, pH 8.9 (tris-barbital buffer 0.035 M). The strip is placed on a bridge of 14 cm. The electrophoretic pathway is g cm long. Samples are applied with the Chemetron microapplicator (0.5 ,ul/5 mm). IO. Remove
the strips while they are still hot and examine
them against
the
light of a 60-100 W bulb at a few cm distance. EXPERIMENTAL
RESULTS
All separated fractions are detected only at the end of the bleaching process. This treatment produces a swelling of the strip and a photomultiplication of the coloration. The pattern in Fig. I is typical of a normal serum. The Cellogel RS pattern is very different from a starch gel or polyacrylamide gel pattern. This is due to the fact that the Cellogel RS pattern does not show an enrichment in the y zone produced by the molecular sieving effect and the hydrophilic action which is typical of the other gels. The bands on Cellogel RS are very sharp in spite of the long migration (II--12 cm), because the porosity of this product decreases from one surface (penetrable by proteins) to the other (impenetrable). In this way diffusion is hindered. Procedures apt to detect lipo-4 and glycoproteins7 are applied if the electrophoretic run is carried out with a two-fold sample application (5 ~1). REFERENCES I 0. SMITHIES, Biochem. J., 61 (1955) 629. 2 S. RAYMOND AND L. WEINTRAUB, Science, 130 (1959) 711. Clin. Chim. Ada,
22 (1968) 475-479
FRACTIO~ATIO~
OF SERUM PROTEINS
ON CELLOGEL
479
3 L. ORNSTEIN AND B. DAVIS,Anti. N. Y. Acad.Sci., 121 (1964) 321,404. 4 B. COLFS AND J.VERHEYDEN,CE~~.C~W. Acta, IZ (1967)470. 5 W. MULLER BEISSENHIRTZAND H. KELLER,C&Z. Chim. Ada, 13 (1966) 95. 6 B. LOMANTO, C. VERGANI AND L. MASCARETTI,Atti Accad. Med. Lombarda, 20 (1965); Clin. Chim. Acta, 15 (1967) 169. 7 M.PAGET AND P.COUSTENOBLE,Ann.Biol.Clin. (Paris), 23 (1~65) Izog.
Clin.Chim. Acta, 22 (1~68)475-479