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Small-scale membrane filter electrophoresis and immuno-electrophoresis
VC)l.. 3 (I’J=jSl
SMALI/-SCALE
MEMBRANE ANI>
FILTER
ELECTROPHOIIESIS
IMMUNO-EI,ECTROPHORESlS
The advantages of an electrophoretic method which permits the separation of small quantities of protein in a short period of time are apparent. The method to be described is simple, rapid, sensitive and economical. It is an adaptation of the membrane filter electrophoresis described a year ago’ and the technique is essential!, the same. Modifications have been introduced and some details of technique revised after a year’s experience. The disadvantages resulting from the use of a brittle, thin, microporous material are very considerably reduced.
Most horizontal electrophoretic tanks have the cathode and anode buffer compartments separated by a glass or perspex plane over which the strip is stretched. As the strips heat up under current, considerable temperature differences develop between the area round the warm centre of the strip and the comparatively cool areas over the buffer compartments. This in turn leads to the occurrence of convection currents inside the tank with their distorting effect on the separation pattern, particularly when a thin, microporous supporting medium is used. With filter paper strips these effects are less noticeable because of their comparatively large fluid content. For small-scale2 and micro-electrophoresis we (KOHN AND O’KELLY) have constructed an electrophoretic tank which appears to be specially suitable for membrane filter electrophoresis, and which has given in our hands consistently satisfactory rcsults. The main feature of this tank is that the buffer compartments occupy the whole width and length of the tank without any dividing planes, thus providing a continuous fluid surface. Convection currents are greatly reduced and a more efficient vapour saturation and cooling system are provided. Perspex strip-holders shaped to the curvature of the bridge hold the strip in position under moderate tension which can be easily adjusted by slight rotation. This type of strip-holder also greatly simplifies the manipulation connected with the placing of the strip in position. The strip-holders arc roughened on the under surface and lined with filter paper which hangs down to reach the buffer, thus providing a connection between the strip and the buffer. It is, therefore, not necessary for the strip itself to reach the buffer and it need be only slightly longer than the bridge gap. It is more convenient to have two strip-holders on each side so as to be able to remove or insert some of the strips independently. Figs. I and z show the detail and dimensions of the tank described above. These dimensions, however, are not critical and can be modified. 1Lkfcrc11crs p. 4.j.J
VOL.
3 (1958)
MEMBRANE
451
FILTER ELECTROPHORESIS
PROCEDURE Supporting
wkedium
Cellulose acetate membrane filter strips 12 x 2.5 cm. The dimensions are not critical and were rather chosen for convenience and economy. Longer or wider strips can be used depending on the bridge size and requirements. Before placing them in the buffer solution the strips should be thoroughly inspected for the presence of imperfections of the material, such as ridges, oblique lines and blotches. If these are present the strip should be discarded. The strip is impregnated by lowering it slowly into the buffer solution or by floating it on the surface of the buffer and letting it soak in from underneath. Quick immersion traps air and creates opaque areas.
Fig. I. Electrophoretic tank for small scale electrophoresis: I. with bridge and 4 strips in position; 2, bridge with one strip-holder in position; 3. strip-holders; 4. strip-holders lined with filter paper (Whatman No. I). Apparatus made by Shandon Scientific Co., to our specifications and from our prototype.
Fig. 2. Diagram
of electrophoretic tank: I. bridge; 2. strip-holder; 5. strip; 6. water absorbent material.
3. filter paper;
4. wick;
A veronal buffer pH 8.6, as recommended by LAURELL, LAURELL AND SKOOG~, without calcium lactate, was used as a routine. This buffer concentration however may be varied. Lower ionic strengths, for instance, will produce wider bands and increase the mobility. The optimal volume of buffer is best determined for each size of tank. The volume determines the difference in levels between the buffer surface and the strip and this, it was found, greatly influences the pattern and mobility. The greater the difference in level the slower the separation and the narrower the band. In the tank of the size as described above the optimal level difference was about 2 cm. Refemces
9. 454
\()I
3 (I(),j$)
pH 8.0 1.0 InAmp. Buffer vol. 250 mf
. I:&.
3. TiIllC,
length
4
:
of run and separation
pattern
in relation
t(~ starting
line
gap of 8 cm the sample is applied about 2-3 cm from the cathode end of the bridge (ix., onequarter to one-third of the distance). The volume of the sample which can bc apphecl is limited and smaller than in filter paper. It can vary from o.ooor ml to about 0.01 ml jo I /(l-lo ,ul). For serum proteins best results wverc obtained with about o OOI 0.003 ml on a I .,5-cm streak. Larger quantities of protein may interfere with the staining and scanning. For immune-electrophoresis, however, when larger quantities of the antigen may be required, up to 0.01 5 ml can be applied. In this case the sample is applied by placing it on to the strip either as a drop or as a transverse streak, and the run is started without waiting for the fiuitl to soak into the supporting medium.
Under conditions as described a current of roo-r5o \; constant at about 1 111.4per strip applied for about ~-2 & h wiil provide a separation pattern of about 5-7 cm length.
Fig. 4. a. Micro-electrophoresis LO ,cg protein in 0.30 111 serum, 2 h, Kigrosin ; I,. Post-albumin fraction?, 15 ,ug and IO jl”g protein. The fusion of the small fraction into the albumin with increasing amount of protein shown at t : c. Pre-albumins in human serum, 2 II, pl’igrosin; d. 3 pcl and 2 pcl serum, z$ h and 1 h, Light Green and Ponceau Rerl-Fuchsin, second specimen macroglobulinacmia ; e. Abnormal haemoglohins, 2 h, 1.50 V, unstained strip. All photographs were obtained by contact-printing from uncleared strips.
VOL.
3 (1958)
~~E~~BRA~E
FILTER
~1,ECTROPHORESIS
453
Staining a& n~asking The concentration of the dyes used should be much lower than that recommended for filter paper. Alcoholic Amidoblack, aqueous Light Green, Azocarmin and Ponceau Red-Fuchsin in S-IO% acetic acid were all used with good results. Staining solutions in 3% trichloroacetic acid, followed by a 5% acetic acid washing bath, appear to be even more suitable for cellulose acetate strips. 0.15% Ponceau S in 3% trichloroacetic acid is aclean, convenient stain and is most easily and rapidly washed out from the background. When alcoholic stains are used one of the washing baths should be of the same alcohol concentration as the staining solutions. The alcoholic bath renders the background colourless and only then should the strip be brought down to an acidwater bath. A final aqueous bath is essential in order to avoid curling and shrinkage. For small quantities of protein and also for the demonstration of pre-albumins Nigrosin*, G. T. Gurr (O.OOZ~-0.005% solution in 2% acetic acid) should be used. With such a solution the background is practically colourless even before washing. One hour’s staining gives satisfactory results but the optima.1 time is best judged by experience. After staining the strips are washed in 2% acetx acrd or tap water. Drying After the last wash, the strip is blotted dry, left for a few minutes at room temperature and then placed between sheets of filter paper under pressure. A photographic print dryer has also been used with good results. Scanning of the cleared strips is performed in the usual manner, using a proportionately smaller slit. Contact prints from stained uncleared strips are very satisfactory. These can be contact printed again to give positive copies. This procedure is particularly useful for the demonstration of abnormal haemoglobins, as only haemoglobin will absorb visible light and the interference of other proteins is thus completely eliminated.
RESULTS
The separation patterns obtained by the small scale method are at least as satisfactory as on the full size strips. They are less subject to band distorsions and less sensitive to small imperfections of material, apparatus and technique (Figs. 3 and 4). This is probably due to a better buffer saturation, diminished evaporation, more efficient cooling and elimination of convection currents. There appears to be a certain optimum lengths of strip and size and type of tank which provides the most favourable conditions for a thin, microporous supporting medium like the cellulose acetate membrane filter material. The method has been found useful, not only for research purposes, but also in routine work, as a large number of sera can be screened in a short time. In combination with a N&rosin stain it enables the separation of very small quantities of protein (Fig. 4a, b) and also the demonstration of pre-albumin fractions (Fig. 4a, c). When only small quantities of protein are used some minor protein fractions become unmasked which with larger amounts of protein are submerged in the diffusion areas of the adjacent large fraction (Fig. 4b). A small fraction somewhat slower than albumin was thus found to be present in low concentration in many human sera. The nature of this fraction is still obscure and is under investigation. A special technique utilizing the particular property of the cellulose acetate medium has been devised for the separation of abnormal haemoglobins and will be reported soon. A clear separation can be achieved in 2-3 h (Fig. 4e). This may prove to be very useful for the screening of large numbers of patients and particularly so for surveying purposes. The method described can be applied to immuno-electrophoresis5, the technique being the same as reported in a previous communication% but on a smaller scale. It is less time-consuming, has the advantage of a considerable saving of the valuable antiserum and the results are in every way as satisfactory as the full size method References p. 454
I cm. When two electrophoretic strips are used on both sides of one antiserum strip, the electrophoretic strips arc cut in half lengthwise but it is also advisable to cut off the part of the strip away from the antiserum so that ahout a ~-cm width is left.
bIy thanks are due to I&. T. O'KELLY for his help and suggestions, to Mr. 1;. OLOPADE for technical assistance and to Mr. A. H. SAILL.-\RII for the photographs.
A smali-scale and micro membrane filter ~~l~!c~l-[)plloretic method is described. Samples ranging from O.OOOI ml up to 0.01 ml (0.1 &lo ~1) with a protein content from about 5 ,ug~ooc ,ug can be separated, giving a neat and distinct pattern. B-e-albumins and a not yet identified postalbumin fraction were clearly demonstrated. Combined with the agnr diffusion method it can be used with advantage for small-scale immune-elcctropl~or~sis with a great saving of antiserum.
I: J. HOEN, C&h. ciiirrc. ilcin, 2 (igj7)207. z J. KOHN, Binckenz.J., 68 (Igj8) IO. 3 C. B. LAURELL, S. LAURBLL AND N. SKOOC, C/ix.CAer?t., z (1950) gg. 1 M. ORTEGA, Nature, 179 (1957) 1086. 5 P. GRABAR AND C. A. WILLIAMS, Biochiw. b’iopkw. Acta, 17 (rgjg) 07. 6 J. Kom, h’&wc, r8o (ro57) qH&
Received
March Ioth, rg,S
SMALI/-SCALE
MEMBRANE ANI>
FILTER
ELECTROPHOIIESIS
IMMUNO-EI,ECTROPHORESlS
The advantages of an electrophoretic method which permits the separation of small quantities of protein in a short period of time are apparent. The method to be described is simple, rapid, sensitive and economical. It is an adaptation of the membrane filter electrophoresis described a year ago’ and the technique is essential!, the same. Modifications have been introduced and some details of technique revised after a year’s experience. The disadvantages resulting from the use of a brittle, thin, microporous material are very considerably reduced.
Most horizontal electrophoretic tanks have the cathode and anode buffer compartments separated by a glass or perspex plane over which the strip is stretched. As the strips heat up under current, considerable temperature differences develop between the area round the warm centre of the strip and the comparatively cool areas over the buffer compartments. This in turn leads to the occurrence of convection currents inside the tank with their distorting effect on the separation pattern, particularly when a thin, microporous supporting medium is used. With filter paper strips these effects are less noticeable because of their comparatively large fluid content. For small-scale2 and micro-electrophoresis we (KOHN AND O’KELLY) have constructed an electrophoretic tank which appears to be specially suitable for membrane filter electrophoresis, and which has given in our hands consistently satisfactory rcsults. The main feature of this tank is that the buffer compartments occupy the whole width and length of the tank without any dividing planes, thus providing a continuous fluid surface. Convection currents are greatly reduced and a more efficient vapour saturation and cooling system are provided. Perspex strip-holders shaped to the curvature of the bridge hold the strip in position under moderate tension which can be easily adjusted by slight rotation. This type of strip-holder also greatly simplifies the manipulation connected with the placing of the strip in position. The strip-holders arc roughened on the under surface and lined with filter paper which hangs down to reach the buffer, thus providing a connection between the strip and the buffer. It is, therefore, not necessary for the strip itself to reach the buffer and it need be only slightly longer than the bridge gap. It is more convenient to have two strip-holders on each side so as to be able to remove or insert some of the strips independently. Figs. I and z show the detail and dimensions of the tank described above. These dimensions, however, are not critical and can be modified. 1Lkfcrc11crs p. 4.j.J
VOL.
3 (1958)
MEMBRANE
451
FILTER ELECTROPHORESIS
PROCEDURE Supporting
wkedium
Cellulose acetate membrane filter strips 12 x 2.5 cm. The dimensions are not critical and were rather chosen for convenience and economy. Longer or wider strips can be used depending on the bridge size and requirements. Before placing them in the buffer solution the strips should be thoroughly inspected for the presence of imperfections of the material, such as ridges, oblique lines and blotches. If these are present the strip should be discarded. The strip is impregnated by lowering it slowly into the buffer solution or by floating it on the surface of the buffer and letting it soak in from underneath. Quick immersion traps air and creates opaque areas.
Fig. I. Electrophoretic tank for small scale electrophoresis: I. with bridge and 4 strips in position; 2, bridge with one strip-holder in position; 3. strip-holders; 4. strip-holders lined with filter paper (Whatman No. I). Apparatus made by Shandon Scientific Co., to our specifications and from our prototype.
Fig. 2. Diagram
of electrophoretic tank: I. bridge; 2. strip-holder; 5. strip; 6. water absorbent material.
3. filter paper;
4. wick;
A veronal buffer pH 8.6, as recommended by LAURELL, LAURELL AND SKOOG~, without calcium lactate, was used as a routine. This buffer concentration however may be varied. Lower ionic strengths, for instance, will produce wider bands and increase the mobility. The optimal volume of buffer is best determined for each size of tank. The volume determines the difference in levels between the buffer surface and the strip and this, it was found, greatly influences the pattern and mobility. The greater the difference in level the slower the separation and the narrower the band. In the tank of the size as described above the optimal level difference was about 2 cm. Refemces
9. 454
\()I
3 (I(),j$)
pH 8.0 1.0 InAmp. Buffer vol. 250 mf
. I:&.
3. TiIllC,
length
4
:
of run and separation
pattern
in relation
t(~ starting
line
gap of 8 cm the sample is applied about 2-3 cm from the cathode end of the bridge (ix., onequarter to one-third of the distance). The volume of the sample which can bc apphecl is limited and smaller than in filter paper. It can vary from o.ooor ml to about 0.01 ml jo I /(l-lo ,ul). For serum proteins best results wverc obtained with about o OOI 0.003 ml on a I .,5-cm streak. Larger quantities of protein may interfere with the staining and scanning. For immune-electrophoresis, however, when larger quantities of the antigen may be required, up to 0.01 5 ml can be applied. In this case the sample is applied by placing it on to the strip either as a drop or as a transverse streak, and the run is started without waiting for the fiuitl to soak into the supporting medium.
Under conditions as described a current of roo-r5o \; constant at about 1 111.4per strip applied for about ~-2 & h wiil provide a separation pattern of about 5-7 cm length.
Fig. 4. a. Micro-electrophoresis LO ,cg protein in 0.30 111 serum, 2 h, Kigrosin ; I,. Post-albumin fraction?, 15 ,ug and IO jl”g protein. The fusion of the small fraction into the albumin with increasing amount of protein shown at t : c. Pre-albumins in human serum, 2 II, pl’igrosin; d. 3 pcl and 2 pcl serum, z$ h and 1 h, Light Green and Ponceau Rerl-Fuchsin, second specimen macroglobulinacmia ; e. Abnormal haemoglohins, 2 h, 1.50 V, unstained strip. All photographs were obtained by contact-printing from uncleared strips.
VOL.
3 (1958)
~~E~~BRA~E
FILTER
~1,ECTROPHORESIS
453
Staining a& n~asking The concentration of the dyes used should be much lower than that recommended for filter paper. Alcoholic Amidoblack, aqueous Light Green, Azocarmin and Ponceau Red-Fuchsin in S-IO% acetic acid were all used with good results. Staining solutions in 3% trichloroacetic acid, followed by a 5% acetic acid washing bath, appear to be even more suitable for cellulose acetate strips. 0.15% Ponceau S in 3% trichloroacetic acid is aclean, convenient stain and is most easily and rapidly washed out from the background. When alcoholic stains are used one of the washing baths should be of the same alcohol concentration as the staining solutions. The alcoholic bath renders the background colourless and only then should the strip be brought down to an acidwater bath. A final aqueous bath is essential in order to avoid curling and shrinkage. For small quantities of protein and also for the demonstration of pre-albumins Nigrosin*, G. T. Gurr (O.OOZ~-0.005% solution in 2% acetic acid) should be used. With such a solution the background is practically colourless even before washing. One hour’s staining gives satisfactory results but the optima.1 time is best judged by experience. After staining the strips are washed in 2% acetx acrd or tap water. Drying After the last wash, the strip is blotted dry, left for a few minutes at room temperature and then placed between sheets of filter paper under pressure. A photographic print dryer has also been used with good results. Scanning of the cleared strips is performed in the usual manner, using a proportionately smaller slit. Contact prints from stained uncleared strips are very satisfactory. These can be contact printed again to give positive copies. This procedure is particularly useful for the demonstration of abnormal haemoglobins, as only haemoglobin will absorb visible light and the interference of other proteins is thus completely eliminated.
RESULTS
The separation patterns obtained by the small scale method are at least as satisfactory as on the full size strips. They are less subject to band distorsions and less sensitive to small imperfections of material, apparatus and technique (Figs. 3 and 4). This is probably due to a better buffer saturation, diminished evaporation, more efficient cooling and elimination of convection currents. There appears to be a certain optimum lengths of strip and size and type of tank which provides the most favourable conditions for a thin, microporous supporting medium like the cellulose acetate membrane filter material. The method has been found useful, not only for research purposes, but also in routine work, as a large number of sera can be screened in a short time. In combination with a N&rosin stain it enables the separation of very small quantities of protein (Fig. 4a, b) and also the demonstration of pre-albumin fractions (Fig. 4a, c). When only small quantities of protein are used some minor protein fractions become unmasked which with larger amounts of protein are submerged in the diffusion areas of the adjacent large fraction (Fig. 4b). A small fraction somewhat slower than albumin was thus found to be present in low concentration in many human sera. The nature of this fraction is still obscure and is under investigation. A special technique utilizing the particular property of the cellulose acetate medium has been devised for the separation of abnormal haemoglobins and will be reported soon. A clear separation can be achieved in 2-3 h (Fig. 4e). This may prove to be very useful for the screening of large numbers of patients and particularly so for surveying purposes. The method described can be applied to immuno-electrophoresis5, the technique being the same as reported in a previous communication% but on a smaller scale. It is less time-consuming, has the advantage of a considerable saving of the valuable antiserum and the results are in every way as satisfactory as the full size method References p. 454
I cm. When two electrophoretic strips are used on both sides of one antiserum strip, the electrophoretic strips arc cut in half lengthwise but it is also advisable to cut off the part of the strip away from the antiserum so that ahout a ~-cm width is left.
bIy thanks are due to I&. T. O'KELLY for his help and suggestions, to Mr. 1;. OLOPADE for technical assistance and to Mr. A. H. SAILL.-\RII for the photographs.
A smali-scale and micro membrane filter ~~l~!c~l-[)plloretic method is described. Samples ranging from O.OOOI ml up to 0.01 ml (0.1 &lo ~1) with a protein content from about 5 ,ug~ooc ,ug can be separated, giving a neat and distinct pattern. B-e-albumins and a not yet identified postalbumin fraction were clearly demonstrated. Combined with the agnr diffusion method it can be used with advantage for small-scale immune-elcctropl~or~sis with a great saving of antiserum.
I: J. HOEN, C&h. ciiirrc. ilcin, 2 (igj7)207. z J. KOHN, Binckenz.J., 68 (Igj8) IO. 3 C. B. LAURELL, S. LAURBLL AND N. SKOOC, C/ix.CAer?t., z (1950) gg. 1 M. ORTEGA, Nature, 179 (1957) 1086. 5 P. GRABAR AND C. A. WILLIAMS, Biochiw. b’iopkw. Acta, 17 (rgjg) 07. 6 J. Kom, h’&wc, r8o (ro57) qH&
Received
March Ioth, rg,S