Journal of Immunological Methods 7 (1975) 39-46
© North-Holland Publishing Company
COMBINATION OF PORE GRADIENT POLYACRYLAMIDE GEL ELECTROPHORESIS AND CROSSED AGAROSE GEL IMMUNOELECTROPHORESIS Jean DAUSSANT and A. SKAKOUN Laboratoire de Physiologie des Organes V~g~taux, C.N.R.S., 92190 Meudon, France
Received 10 September 1974, accepted 24 September 1974 A technique is described which combines electrophoresis in pore gradient polyacrylamide gel with crossed electrophoresis in an agarose gel containing an immune serum. This technique enables the study of antigenic relationships between constituents which differ in their molecular size. An example is provided with different oligomeric forms of purified #-amylase.
1. Introduction Tile immunoelectrophoretic analysis (Grabar and Williams, 1953) which has allowed considerable progress in protein studies gave the start to many developments. The method has proved its efficiency in qualitative studies and was further developed in order to provide quantitative data: two-dimensional electrophoresis of antigens with antibody-containing buffer (Ressler, 1960) and antigen-antibody crossed electrophoresis (Laurell, 1966). These methods were further elaborated and their numerous refinements and applications were reviewed (Axelsen et al., 1973). Moreover, the use of polyacrylamide gel for an initial electrophoresis of the proteins followed by a crossed immunoelectrophoresis in agarose gel combined the high separation power of the polyacrylamide gel with the quantitative capacity of the crossed agarose gel immunoelectrophoresis (Johansson and Stenflo, 1971; Giebel and Saechtling, 1973). In all the preceding techniques, when a protein separation is completed before the antigen antibody reaction, the separation is based on the charges of the proteins at the pH chosen for the electrophoresis. The characterization nevertheless does not provide any information on possible structural relationships between the separated constituents. In pore gradient polyacrylamide gel, however, (Margolis and Kenrick, 1968), and under certain conditions (Skakoun and Daussant, 1972), the electrophoretically separated constituents are characterized by their molecular weight. Such data may suggest structural relationships between certain of the 39
40
J. Daussant, A. Skakoun, Combination of electrophoretic techniques
separated constituents: some of them may occur as polymers of others. Thus it seemed particularly interesting to combine an immunochemical analysis with this type of electrophoresis. Such a combination could be expected to deliver in a single experiment two sorts of information molecular weight and antigenicity both of interest for a structural comparison of different constituents, particularly in studies concerned with oligomeric forms of proteins and enzymes. For the elaboration of the technique, we used purified barley/3-amylase. It was proved that this enzyme exists in several oligomeric forms which were transformed into the smallest one under the action of reducing agents (Niku-Paavola et al., 1973).
2. Materials and methods
2.1. Antigen
/3-Amylase from barley (Pirkka, a Finnish 6-row variety) purified by Visuri and Nummi (1972) was used in this study. 2.2. I m m u n e serum The immune serum was a pool of sera obtained from three rabbits immunized with total barley protein extracts (Daussant, 1966).
2.3. Pore gradient polyacrylamide gel electrophoresis
Tile method of Margolis and Kenrick (1968) was used under the conditions previously described for barley /3-amylase (Niku-Paavola et al., 1973). The polyacrylamide gel used had an acrylamide concentration ranging from 4% (side of the gel on which proteins are deposited) to 30%, the electrophoresis being conducted in a Tris glycine buffer, pH 8.4. Human serum albumin and different amounts of /3-amylase, dissolved in the electrophoresis buffer containing 20% saccharose, were deposited in different channels on the top of the gel: 15 H1 of 10 mg/ml human serum albumin were applied for establishing a molecular size reference scale, 45/Jl of 7 mg/ml/3-amylase was used for subsequent immunochemical analysis and 10/al of 0.7 mg/ml /3-amylase was employed for amylase characterisation after electrophoresis. Electrophoresis was conducted at 3°C for 62 hr in order to reach the conditions required for a valuable molecular size determination (Skakoun and Daussant, 1972). After the electrophoresis was completed, the gel was divided into two thinner plates. Slabs were then cut according to the direction of electrophoresis migration in the thicker half of the gel plate.
+
embedding an
agarose gel
~ +
50
79
o 60
_
[: 2 0
10
0
~ 67
Molecular
¥/
e
of H S A
, , , ~ 201268 (103)
weight
, 134
o~
+
?ig. 1. Electrophoretic and immunoelectrophoretic analysis of purified ~3-amylase. A and E: Combination of pore gradient polyacrylamide gel dectrophoresis as reported in B and crossed agarose gel immunoelectrophoresis. /3-Amylase characterization. In E the agarose gels contain 3.03% mercaptoethanol. Note the presence of residual ¢~-amylase activity in the polyacrylamide slab in A, and the differences between the mmunoelectropherograms in A and E. First electrophoresis: eleetrophoresis performed in the pore gradient polyacrylamide gel. Second electro)horesis: crossed electrophoresis in agarose gel containing the immune serum; B: pore gradient polyacrylamide gel electrophoresis of ~-amylase. ~-amylase characterization; C: Relationship between molecular weight of proteins and migration distance. Human serum albumin served for ,'stablishing the reference scale. Arrows indicate the migration distance corresponding to the ~-amylase constituents detected in B.
4-
E
30% ~
acrylamide gel slab
the
2nd electrophoresis
immune serum
agarose gel containing
4%~
~a
3
?
~a
42
.L Daussant, A. Skakoun, Combination oJ' eleetrophoretic techniques
Tire slab containing the human serum albumin was stained with Amido Black (fig. 1D). Tire stab containing the smaller amount of/3-amylase was incubated 5 hr in a 2% soluble starch solution and stained with iodine (Daussant el al., 1965) (fig. 1B). The slabs containing tire higher amounts of/3-amylase were used for tire subsequent imnmnochemical analysis.
2.4. Combined pore gradient polyacrvlarnide gel eIectrophoresis and agarose gel crossed immunoelectrophoresis A pore gradient polyacrylamide gel slab (3 mm wide) containing the previously separated ~-amylase was first washed 1 hr under stirring in 0.025 M veronal buffer, pfl 8.6. This slab was then embedded in a 1% agarose gel prepared in the same buffer (fig. 1A). Another agarose gel containing 10% immune serum was deposited against the embedding gel*. Electrophoresis was conducted under 5 V/cm during 21 hr at 3°C. A glass plate was placed at 1 2 mm above the gel in order to prevent the gel from drying. The plate was washed 2 hr with 8.5 g/1 NaCI solution buffered with veronal and was then incubated 45 min in a 1% soluble starch solution in 0.1 M phosphate buffer, pH 6.0, containing 0.03% nrercaptoethanol, rinsed twice in tapwater and stained with iodine (fig. 1A). Another experiment was conducted under the same conditions except that the buffer serving for washing the polyacrylamide slab and for preparing the agarose gel contained 0.03% mercaptoethanol (fig. 1E).
3. Results and discussion
Tile electrophoresis in pore gradient polyacrylamide gel was run without particular difficulties under conditions previously described (Skakoun and Daussant, 1972; Niku-Paavola et al., 1973). The crossed electrophoresis involving different gels, though simple in principle, encountered nevertheless several technical difficulties resulting in poor immunoelectrophoretic patterns or even making impossible the analysis. Thus, very early alter the crossed electrophoresis started, drops appeared on the agarose gel all along the anodic side of the polyacrylamide gel slab. This was probably at the origin of badly defined patterns, tile protein present in the gel being sucked in by such drops. Furthermore, when a current of 5 V/cm was applied, the polyacrylamide gel slab began to separate from the agarose gel, on its cathodic side, and this became evident already after 4 hr of electrophoresis. The split always began to occur at the less dense end of the polyacrylamide gel slab. Such a phenomenon has been ascribed to electroendosmosis differences displayed between two different gels (Johansson and * The agarose used in this study was a special agarose batch no. 50 786 produced by Industrie Biologique Francaise.
J. Daussant, A. Skakoun, Combination of electrophoretic techniques
43
Stenflo, 1971). In order to overcome these difficulties we used a special agarose batch which showed a smaller electroendosmotic current than the commonly available agarose. The difficulties could further be reduced by washing the polyacrylamide gel slab in the buffer used for preparing the agarose gel. We checked that during this washing the loss of enzyme was minimal: no difference could be detected between electrophoretic patterns of 13-amylase obtained from washed and unwashed polyacrylamide gel slabs. With the fi-amylase preparation employed and under the conditions used in the first electrophoresis, a series of 6 constituents bearing the/Lamylase activity were detected (fig. 1B). The values of molecular weight found for the three smallest constituents were 54,000 105,000 and 140,000 daltons (fig. 1C). Taking into account the approximation concerning these values~ the results suggest that these constituents may correspond to monomeric, dimeric, trimeric forms of the enzyme as already reported (Niku-Paavola et al., 1973). On the immunoelectropherogram three distinct peaks bearing the ~3-amylaseactivity were identified; they correspond, because of their location, to the three smallest constituents of the enzyme (fig. 1A). Further I3-amylase constituents were antigenically detected as shoulders occurring on the cathodic side of the third peak. Two observations may be outlined concerning these results: 1) The totality of enzymes corresponding to the oligomeric forms have not been extracted from the polyacrylamide gel slab, although the conditions used for the crossed immunoelectrophoresis were sufficient for extracting all the molecules corresponding to the smallest forms of the enzyme from tire slab (fig. IA). 2) On fig. 1A, there is a semi-identity reaction between peak 2 and peak 3 (arrow). This result might indicate that the enzyme in the dimeric form has antigenic sites which are hidden in the more elaborated oligomeric form. Furthermore, the cathodic side of the second peak seems to give an identity reaction with a precipitin band forming a small peak just below peak 3. This suggests the existence of several antigenic trimeric forms of the enzyme; thus, the trimeric forms could correspond to hetero-oligomers. It is worth noticing that a semi-identity reaction also occurs between the first and the second peak. Knowing that the oligomeric forms of the enzyme were transformed into the monomers under the action of reducting agents (Niku-Paavola et al., 1973), it was tempting to compare the antigenic relationships between /J-amylase constituents which were first separated according to their molecular size and then reduced to their monomeric forms. In the first part of the experiment, electrophoresis in pore gradient polyacrylamide gel was performed under the same conditions as just reported. The crossed immunoelectrophoresis was also carried out under the same conditions as already reported with the only difference that the buffer for washing the polyacrylamide gel slab and the agarose gel contained both mercaptoethanol 0.03%. During the crossed electrophoresis the oligomeric forms were reduced to smaller ones. This probably began already in the polyacrylamide gel slab during the washing in the veronal buffer containing 0.03% mercaptoethanol. The results are
44
J. Daussant, A. Skakoun. Combination of electrophoretic techniques
reported on fig. 1E -- comparison between fig. 1A and fig. 1E calls for several observations: 1) When using a reducing agent, all the /3-amylase constituents were extracted from the polyacrylamide gel slab. The use of this technique for quantitative analysis actually implies that all the molecules are participating in the antigen-antibody reaction; thus, in the present case, the use of a reducing agent prevents the retention of the enzyme in the polyacrylamide gel slab and might enable quantitative determinations. 2) The use of a reducing agent furthermore resulted in the disappearance of semi-identity reactions observed when the experiment was run without a reducing agent. Thus, an evaluation of the proportions in which the enzyme exists in the different forms would be possible such a determination actually requires that there be identity reactions between the enzymes previously separated by the first electrophoresis. 3) The fact that peaks 2 and 3 are higher in the experiment with mercaptoethanol is not surprising since more enzyme corresponding to these peaks are extracted from the polyacrylamide gel slab. Moreover, the difference in the sizes of the peak may also be due to the fact that the enzyme molecules present more antigenic sites in a monomeric than in an oligomeric form. 4) Lastly, there are two points which call for further studies: peak 3 (fig. 1E) seems to form a double band which is visible at best between peak 2 and peak 3. This result would suggest the existence of two different monomeric antigenic units building up the trimeric form of the enzyme. The shoulders of the cathodic side of peak 3 (fig. I A) disappeared rather than being higher when the experiment was run with mercaptoethanol. 4. Conclusions The combination of pore gradient polyacrylamide gel electrophoresis with crossed agarose gel immunoelectrophoresis presents some technical difficulties which nevertheless can be overcome under certain conditions which we have determined. In a single experiment this method allows one to obtain information on the molecular size and on the antigenic character of proteins. Such results seem particularly interesting in studies concerned with enzymatic polymorphism, identification of homo- or hetero-oligomeric forms of proteins, particularly enzymes. Under certain conditions the method might provide data concerning the quantitative distribution of the enzyme in different oligomeric forms.
Acknowledgements The authors thank Miss C. Mayer for technical assistance. The authors thank the 'Syndicats de la Malterie et de la Brasserie Francaise' and the 'Centre Technique de la Malterie et Brasserie, Nancy' for their support.
J. Daussant, A. Skakoun, Combination of eleetrophoretic techniques
45
References Axelsen, N.H., J. Kr¢ll and B. Weeke, 1973, Scand. J. Immunol. 2, suppl. 1. Daussant, J., 1966, Biotechnique 4, 1. Daussant, J., P. Grabar and M. Nummi, 1965, in: Eur. Brewery Cony. Proc. 10th Congr. Stockholm, 62, Elsevier (Amsterdam). Giebel, W. and H. Saechtling, 1973, Hoppe Seyler's Z. Physiol. Chem. 354,673. Grabar, P. and C.A. Williams, 1953, Biochim. Biophys. Acta 10, 193. Johansson, B.G. and J. Stenflo, 1971, Anal. Biochem. 40,232. Laurell, C.B., 1966, Anal. Biochem. 15, 45. Margolis, J. and K.G. Kenrick, 1968, Anal. Biochem. 25,347. Niku-Paavola, M.L., A. Skakoun, M. Nummi and J. Daussant, 1973, Biochim. Biophys. Acta 322, 181. Ressler, N., 1960, Clin. Chim. Acta 5,795. Skakoun, A. and J. Daussant, 1972, Bios. 11,503. Visuri,K. and M. Nummi, 1972, Eur. J. Biochem. 28,555.
© North-Holland Publishing Company
COMBINATION OF PORE GRADIENT POLYACRYLAMIDE GEL ELECTROPHORESIS AND CROSSED AGAROSE GEL IMMUNOELECTROPHORESIS Jean DAUSSANT and A. SKAKOUN Laboratoire de Physiologie des Organes V~g~taux, C.N.R.S., 92190 Meudon, France
Received 10 September 1974, accepted 24 September 1974 A technique is described which combines electrophoresis in pore gradient polyacrylamide gel with crossed electrophoresis in an agarose gel containing an immune serum. This technique enables the study of antigenic relationships between constituents which differ in their molecular size. An example is provided with different oligomeric forms of purified #-amylase.
1. Introduction Tile immunoelectrophoretic analysis (Grabar and Williams, 1953) which has allowed considerable progress in protein studies gave the start to many developments. The method has proved its efficiency in qualitative studies and was further developed in order to provide quantitative data: two-dimensional electrophoresis of antigens with antibody-containing buffer (Ressler, 1960) and antigen-antibody crossed electrophoresis (Laurell, 1966). These methods were further elaborated and their numerous refinements and applications were reviewed (Axelsen et al., 1973). Moreover, the use of polyacrylamide gel for an initial electrophoresis of the proteins followed by a crossed immunoelectrophoresis in agarose gel combined the high separation power of the polyacrylamide gel with the quantitative capacity of the crossed agarose gel immunoelectrophoresis (Johansson and Stenflo, 1971; Giebel and Saechtling, 1973). In all the preceding techniques, when a protein separation is completed before the antigen antibody reaction, the separation is based on the charges of the proteins at the pH chosen for the electrophoresis. The characterization nevertheless does not provide any information on possible structural relationships between the separated constituents. In pore gradient polyacrylamide gel, however, (Margolis and Kenrick, 1968), and under certain conditions (Skakoun and Daussant, 1972), the electrophoretically separated constituents are characterized by their molecular weight. Such data may suggest structural relationships between certain of the 39
40
J. Daussant, A. Skakoun, Combination of electrophoretic techniques
separated constituents: some of them may occur as polymers of others. Thus it seemed particularly interesting to combine an immunochemical analysis with this type of electrophoresis. Such a combination could be expected to deliver in a single experiment two sorts of information molecular weight and antigenicity both of interest for a structural comparison of different constituents, particularly in studies concerned with oligomeric forms of proteins and enzymes. For the elaboration of the technique, we used purified barley/3-amylase. It was proved that this enzyme exists in several oligomeric forms which were transformed into the smallest one under the action of reducing agents (Niku-Paavola et al., 1973).
2. Materials and methods
2.1. Antigen
/3-Amylase from barley (Pirkka, a Finnish 6-row variety) purified by Visuri and Nummi (1972) was used in this study. 2.2. I m m u n e serum The immune serum was a pool of sera obtained from three rabbits immunized with total barley protein extracts (Daussant, 1966).
2.3. Pore gradient polyacrylamide gel electrophoresis
Tile method of Margolis and Kenrick (1968) was used under the conditions previously described for barley /3-amylase (Niku-Paavola et al., 1973). The polyacrylamide gel used had an acrylamide concentration ranging from 4% (side of the gel on which proteins are deposited) to 30%, the electrophoresis being conducted in a Tris glycine buffer, pH 8.4. Human serum albumin and different amounts of /3-amylase, dissolved in the electrophoresis buffer containing 20% saccharose, were deposited in different channels on the top of the gel: 15 H1 of 10 mg/ml human serum albumin were applied for establishing a molecular size reference scale, 45/Jl of 7 mg/ml/3-amylase was used for subsequent immunochemical analysis and 10/al of 0.7 mg/ml /3-amylase was employed for amylase characterisation after electrophoresis. Electrophoresis was conducted at 3°C for 62 hr in order to reach the conditions required for a valuable molecular size determination (Skakoun and Daussant, 1972). After the electrophoresis was completed, the gel was divided into two thinner plates. Slabs were then cut according to the direction of electrophoresis migration in the thicker half of the gel plate.
+
embedding an
agarose gel
~ +
50
79
o 60
_
[: 2 0
10
0
~ 67
Molecular
¥/
e
of H S A
, , , ~ 201268 (103)
weight
, 134
o~
+
?ig. 1. Electrophoretic and immunoelectrophoretic analysis of purified ~3-amylase. A and E: Combination of pore gradient polyacrylamide gel dectrophoresis as reported in B and crossed agarose gel immunoelectrophoresis. /3-Amylase characterization. In E the agarose gels contain 3.03% mercaptoethanol. Note the presence of residual ¢~-amylase activity in the polyacrylamide slab in A, and the differences between the mmunoelectropherograms in A and E. First electrophoresis: eleetrophoresis performed in the pore gradient polyacrylamide gel. Second electro)horesis: crossed electrophoresis in agarose gel containing the immune serum; B: pore gradient polyacrylamide gel electrophoresis of ~-amylase. ~-amylase characterization; C: Relationship between molecular weight of proteins and migration distance. Human serum albumin served for ,'stablishing the reference scale. Arrows indicate the migration distance corresponding to the ~-amylase constituents detected in B.
4-
E
30% ~
acrylamide gel slab
the
2nd electrophoresis
immune serum
agarose gel containing
4%~
~a
3
?
~a
42
.L Daussant, A. Skakoun, Combination oJ' eleetrophoretic techniques
Tire slab containing the human serum albumin was stained with Amido Black (fig. 1D). Tire stab containing the smaller amount of/3-amylase was incubated 5 hr in a 2% soluble starch solution and stained with iodine (Daussant el al., 1965) (fig. 1B). The slabs containing tire higher amounts of/3-amylase were used for tire subsequent imnmnochemical analysis.
2.4. Combined pore gradient polyacrvlarnide gel eIectrophoresis and agarose gel crossed immunoelectrophoresis A pore gradient polyacrylamide gel slab (3 mm wide) containing the previously separated ~-amylase was first washed 1 hr under stirring in 0.025 M veronal buffer, pfl 8.6. This slab was then embedded in a 1% agarose gel prepared in the same buffer (fig. 1A). Another agarose gel containing 10% immune serum was deposited against the embedding gel*. Electrophoresis was conducted under 5 V/cm during 21 hr at 3°C. A glass plate was placed at 1 2 mm above the gel in order to prevent the gel from drying. The plate was washed 2 hr with 8.5 g/1 NaCI solution buffered with veronal and was then incubated 45 min in a 1% soluble starch solution in 0.1 M phosphate buffer, pH 6.0, containing 0.03% nrercaptoethanol, rinsed twice in tapwater and stained with iodine (fig. 1A). Another experiment was conducted under the same conditions except that the buffer serving for washing the polyacrylamide slab and for preparing the agarose gel contained 0.03% mercaptoethanol (fig. 1E).
3. Results and discussion
Tile electrophoresis in pore gradient polyacrylamide gel was run without particular difficulties under conditions previously described (Skakoun and Daussant, 1972; Niku-Paavola et al., 1973). The crossed electrophoresis involving different gels, though simple in principle, encountered nevertheless several technical difficulties resulting in poor immunoelectrophoretic patterns or even making impossible the analysis. Thus, very early alter the crossed electrophoresis started, drops appeared on the agarose gel all along the anodic side of the polyacrylamide gel slab. This was probably at the origin of badly defined patterns, tile protein present in the gel being sucked in by such drops. Furthermore, when a current of 5 V/cm was applied, the polyacrylamide gel slab began to separate from the agarose gel, on its cathodic side, and this became evident already after 4 hr of electrophoresis. The split always began to occur at the less dense end of the polyacrylamide gel slab. Such a phenomenon has been ascribed to electroendosmosis differences displayed between two different gels (Johansson and * The agarose used in this study was a special agarose batch no. 50 786 produced by Industrie Biologique Francaise.
J. Daussant, A. Skakoun, Combination of electrophoretic techniques
43
Stenflo, 1971). In order to overcome these difficulties we used a special agarose batch which showed a smaller electroendosmotic current than the commonly available agarose. The difficulties could further be reduced by washing the polyacrylamide gel slab in the buffer used for preparing the agarose gel. We checked that during this washing the loss of enzyme was minimal: no difference could be detected between electrophoretic patterns of 13-amylase obtained from washed and unwashed polyacrylamide gel slabs. With the fi-amylase preparation employed and under the conditions used in the first electrophoresis, a series of 6 constituents bearing the/Lamylase activity were detected (fig. 1B). The values of molecular weight found for the three smallest constituents were 54,000 105,000 and 140,000 daltons (fig. 1C). Taking into account the approximation concerning these values~ the results suggest that these constituents may correspond to monomeric, dimeric, trimeric forms of the enzyme as already reported (Niku-Paavola et al., 1973). On the immunoelectropherogram three distinct peaks bearing the ~3-amylaseactivity were identified; they correspond, because of their location, to the three smallest constituents of the enzyme (fig. 1A). Further I3-amylase constituents were antigenically detected as shoulders occurring on the cathodic side of the third peak. Two observations may be outlined concerning these results: 1) The totality of enzymes corresponding to the oligomeric forms have not been extracted from the polyacrylamide gel slab, although the conditions used for the crossed immunoelectrophoresis were sufficient for extracting all the molecules corresponding to the smallest forms of the enzyme from tire slab (fig. IA). 2) On fig. 1A, there is a semi-identity reaction between peak 2 and peak 3 (arrow). This result might indicate that the enzyme in the dimeric form has antigenic sites which are hidden in the more elaborated oligomeric form. Furthermore, the cathodic side of the second peak seems to give an identity reaction with a precipitin band forming a small peak just below peak 3. This suggests the existence of several antigenic trimeric forms of the enzyme; thus, the trimeric forms could correspond to hetero-oligomers. It is worth noticing that a semi-identity reaction also occurs between the first and the second peak. Knowing that the oligomeric forms of the enzyme were transformed into the monomers under the action of reducting agents (Niku-Paavola et al., 1973), it was tempting to compare the antigenic relationships between /J-amylase constituents which were first separated according to their molecular size and then reduced to their monomeric forms. In the first part of the experiment, electrophoresis in pore gradient polyacrylamide gel was performed under the same conditions as just reported. The crossed immunoelectrophoresis was also carried out under the same conditions as already reported with the only difference that the buffer for washing the polyacrylamide gel slab and the agarose gel contained both mercaptoethanol 0.03%. During the crossed electrophoresis the oligomeric forms were reduced to smaller ones. This probably began already in the polyacrylamide gel slab during the washing in the veronal buffer containing 0.03% mercaptoethanol. The results are
44
J. Daussant, A. Skakoun. Combination of electrophoretic techniques
reported on fig. 1E -- comparison between fig. 1A and fig. 1E calls for several observations: 1) When using a reducing agent, all the /3-amylase constituents were extracted from the polyacrylamide gel slab. The use of this technique for quantitative analysis actually implies that all the molecules are participating in the antigen-antibody reaction; thus, in the present case, the use of a reducing agent prevents the retention of the enzyme in the polyacrylamide gel slab and might enable quantitative determinations. 2) The use of a reducing agent furthermore resulted in the disappearance of semi-identity reactions observed when the experiment was run without a reducing agent. Thus, an evaluation of the proportions in which the enzyme exists in the different forms would be possible such a determination actually requires that there be identity reactions between the enzymes previously separated by the first electrophoresis. 3) The fact that peaks 2 and 3 are higher in the experiment with mercaptoethanol is not surprising since more enzyme corresponding to these peaks are extracted from the polyacrylamide gel slab. Moreover, the difference in the sizes of the peak may also be due to the fact that the enzyme molecules present more antigenic sites in a monomeric than in an oligomeric form. 4) Lastly, there are two points which call for further studies: peak 3 (fig. 1E) seems to form a double band which is visible at best between peak 2 and peak 3. This result would suggest the existence of two different monomeric antigenic units building up the trimeric form of the enzyme. The shoulders of the cathodic side of peak 3 (fig. I A) disappeared rather than being higher when the experiment was run with mercaptoethanol. 4. Conclusions The combination of pore gradient polyacrylamide gel electrophoresis with crossed agarose gel immunoelectrophoresis presents some technical difficulties which nevertheless can be overcome under certain conditions which we have determined. In a single experiment this method allows one to obtain information on the molecular size and on the antigenic character of proteins. Such results seem particularly interesting in studies concerned with enzymatic polymorphism, identification of homo- or hetero-oligomeric forms of proteins, particularly enzymes. Under certain conditions the method might provide data concerning the quantitative distribution of the enzyme in different oligomeric forms.
Acknowledgements The authors thank Miss C. Mayer for technical assistance. The authors thank the 'Syndicats de la Malterie et de la Brasserie Francaise' and the 'Centre Technique de la Malterie et Brasserie, Nancy' for their support.
J. Daussant, A. Skakoun, Combination of eleetrophoretic techniques
45
References Axelsen, N.H., J. Kr¢ll and B. Weeke, 1973, Scand. J. Immunol. 2, suppl. 1. Daussant, J., 1966, Biotechnique 4, 1. Daussant, J., P. Grabar and M. Nummi, 1965, in: Eur. Brewery Cony. Proc. 10th Congr. Stockholm, 62, Elsevier (Amsterdam). Giebel, W. and H. Saechtling, 1973, Hoppe Seyler's Z. Physiol. Chem. 354,673. Grabar, P. and C.A. Williams, 1953, Biochim. Biophys. Acta 10, 193. Johansson, B.G. and J. Stenflo, 1971, Anal. Biochem. 40,232. Laurell, C.B., 1966, Anal. Biochem. 15, 45. Margolis, J. and K.G. Kenrick, 1968, Anal. Biochem. 25,347. Niku-Paavola, M.L., A. Skakoun, M. Nummi and J. Daussant, 1973, Biochim. Biophys. Acta 322, 181. Ressler, N., 1960, Clin. Chim. Acta 5,795. Skakoun, A. and J. Daussant, 1972, Bios. 11,503. Visuri,K. and M. Nummi, 1972, Eur. J. Biochem. 28,555.
