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The construction and use of a small and inexpensive apparatus for filter paper electrophoresis
390
voL. 2 (1957)
CLINICA CHIMICA ACTA
THE CONSTRUCTION APPARATUS
AND USE OF A SMALL AND INEXPENSIVE
FOR FILTER
F. RAPPAPORT,
Beilinson Hospital.
PAPER
F. EICHHORN
Petah Tikva, and Governmental
ELECTROPHORESIS
AND
J. FISCHL
Hospital. Assaf Harofe, Zrifin (Israel)
Today, filter paper electrophoresis has become an everyday routine work for progressive laboratories. However, the apparatus made by various commercial firms is very expensive and, therefore, not available for laboratories working on a low budget. To overcome financial difficulties, we have constructed an apparatus from cheap and easily obtainable material. In principle, our apparatus is similar to others, with the exception that the electrodes are placed inside and under the paper-supporting bridge, giving the advantage of greatly reduced size over the other type of apparatus. The results obtained by our apparatus are identical to those obtained with the usual types of instruments. Its much smaller size makes electrophoresis in restricted spaces more convenient, ~.g., electrophoresis done under refrigeration, or in small and crowded laboratories. The purpose of this paper is to give a detailed description of the construction of the apparatus and of methods for its use. CONSTRUCTION
OF APPARATUS
The box to be used, made of chloroform-soluble material and obtainable at hardware stores, should have these dimensions: wall thickness 1.2-1.5 mm; length 19.5 cm, width 9.5 cm, and depth 6.0 cm (inner measurements). Four plates (p), 4 x 9.45 cm, are cut out of a sheet of perspex. Two of these plates are inserted perpendicular to the floor and at right angles to the length, each at I cm distance from the midline. The plates, and all additional parts in the apparatus, are glued to the box by perspex material that has been dissolved in chloroform. The 2 cm space in the midsection between the two plates is completely filled with a melted beeswax-paraffin wax (I : I) mixture (w) . (This prevents leakage and short circuit of the system.) Each of the two remaining plates is pierced 20-25 times within a radius of 1.0-1.5 cm from the center with a red-hot needle. These plates are then inserted parallel with the two others, dividing the box into four open compartments (cf. Fig. I). After fixing the plates into their proper place with chloroform-perspex solution, four little squares of IO x 15 mm (s), cut preferably from colored plastic material, are glued to both sides of the plates 7 mm from the upper edge. They serve as marks for the buffer level, and facilitate removal of surplus buffer. Two holes (h), 4 mm in diam., are drilled on one side of the box for the electrodes. They are made at 3.0 cm distance from the center, and at 2.7 cm from the bottom of the box. Two electrodes are made from copper screws about 6 cm long, 4 mm diam., and without head. At one end of each screw a 1.6 cm deep cut is made, and the same side of the screw is filed smooth and someReferences p. 396
VOL.
2 (1957)
APPARATUS FOR FILTER PAPER ELECTROPHORESIS
391
what conical at the end, in order to serve as electrical connector. About 3 mm from the tip of the other end of the screw, a small hole is bored through the metal, and a piece of platinum wire, 4 cm long, is inserted in such a way that the ends of the wire are protruding in a fork-like manner. These ends are then cleaned with HCI, and the wire is welded to the rods with welding alloy. The electrodes are inserted in the box through the holes, with connecting ends protruding 2 cm outside. Two small
19.5
Fig. I. Schematic
representation
of the apparatus.
copper bolts are then screwed on from both sides, fixing the electrodes firmly to the wall of the box. To isolate the bolts, small pieces of perspex plates are glued over them from both sides. To isolate the copper electrode supports from contact with the buffer solution, rubber tubing is used and must cover all the copper surfaces inside the box. Perspex solution in chloroform serves well, both for glueing tubing to metal and for making an isolating film on the copper surface. The rubber tube is pierced at the proper place, and the platinum wire ends are drawn out through the puncture. The end of the tube is closed by a glass plug that is also glued in. To shield the electrodes, two plates of I mm thick perspex material 6.5 x 3.5 cm (c), are bent at 90’ in such a way that one wing is about 2.5 cm and the other 4 cm long. They are glued to the bottom of the box, the longer side running parallel to the compartment-dividing plates, half-way between plate and electrode, the shorter shield wing circling the electrode in the direction of the wax dividing wall. Two small holes of 3 mm diam. (0) are cut into the box walls on each side of the electrode compartment. They are 3.7 cm from the bottom of the box. Rubber tubes of 3 mm length, with a diam. just fitting the holes, are inserted, one end just a few mm inside the box, the other bent upwards, fixed to the outside of the box by a piece of perspex plate. They serve as outlets for the gases generated during electrophoresis. The combined electrode cover-paper support is prepared in the following way : a 2 mm thick perspex plate of the required width (9.45 cm) is inserted into the box, its length being sufficient to cover both electrode compartments (13 cm). When it is inserted, only the two outer compartments are uncovered. This plate is not glued in, References
p. 396
392
F. RAPPAPORT,
F. EICHHORN,
J. FISCHL
VOL. 2
(1957)
but remains a removable part. When inserting it, a piece of polyethylene wrapping material is placed underneath the perspex plate, covering it completely and overlapping upwards 0.5 cm on each side. This ensures air-tightness of the electrode chambers. To prevent the filter paper from sagging during electrophoresis, a device must be made to support the paper strips. Toy racing cars of plastic material will suffice for this. They should be I cm high and z cm long, resting on a firm base. The drivers’
Fig. 2. Photographic
picture of the apparatus.
heads served excellently for supporting the paper strips. Thirty-six of these cars, divided evenly into two sets of three rows each, are glued to the electrode covering plate. Spaces between the rows are about 1.2 cm, and that between the two sets is about 2.2 cm. The paper-holder bridge is made of 4 mm-thick perspex glass. It is 9.4 cm wide, 18 cm long, and 5.5 cm high. The plane on which the paper is supported is 2 cm wide. To ensure good closure of the apparatus, an elastic plastic band (used to insulate electric wires) is glued to the upper edge of the box, carefully connecting the ends with bees’ wax-paraffin mixture. To cover the apparatus a thick (5 mm) glass plate of sufficient size is used. A transformer-rectifier was built in our local electrical workshop. It is equipped with a voltage meter and with a pilot lamp. It has connections for three sets of apparatuses, and is able to supply six to eight more sets if required. No additional apparatus is needed that is not to be found in the usual standard laboratory equipment, e.g., centrifuge, photoelectric calorimeter, pipettes, test tubes, etc. Rejerences fi 396
VOL. 2 (1957)
APPARATUS FOR FILTER PAPER ELECTROPHORESIS
393
METHODS
Reagents I. 2.
3. 4. 5. 6.
9.8 g
sodium barbital, 6.5 g sodium acetate, 0.1 g sodium azide are made up with water to I I; N/I HCl (& IO ml) is added to adapt the pH to 8.6”. 3”/0 sulfosalicylic acid in water. Dye solution: dissolve 0.8 g light green SF1 and 15 g sulfosalicylic acid in 500 ml distilled water. Decolorising solution: acetic acid 2:; soln. in tap zater. Sodium hydroxide X/so, containing 0.27; human serum. Formalin C.P.
Fill apparatus with buffer solution up to the levelling mark in all four compartments, and remove surplus by a water suction pump or with a syringe. with the cover plate, placing polyethylene paper 2. Cover electrode compartments underneath (see above). 3. Fix two strips of Whatman No. r, or Elphor filter paper, 4 x 28 cm, on the paper support by moistening the ends, and stretch tightly. Insert support into apparatus and cover system with cover plate; this should be greased with a thin layer of silicone grease before closing. To ensure airtightness it is advisable to weight down the lid. The whole apparatus should be covered with a cloth to prevent influence of external temperature changes during the electrophoresis. 4. Switch on current of 8o-go V until strips become saturated with buffer, and leave under current for IO more min. 5 For application of the serum on the paper strips, prepare narrow paper ribbons2 from Whatman No. I paper. They should be 2.8 cm x 2 mm, with a 3 mm-folded end on one side. Hold the folded end of one paper ribbon with a plastic forceps, soak it with serum, and place on the corresponding paper strip at the cathode side at about 2 cm from the edge of the paper support. 6. Cover apparatus and leave under current for 15 min. Remove application strips and cover again. 87: Leave under current for 14-16 h. I.
After the strips have been removed from the apparatus, they are air-dried and placed in the sulfosalicylic solution (Reagent 2) for 15 min, in the dye solution for 30 min (Reagent 31, and finally washed with reagent No. 4 until the paper background is completely decolorised. They are then air-dried (see Fig. 3). * When
exactly (NaN,);
Referestces
no sodium barbital C.P. is available, the following buffer may be used, which gives the same results: 8.8. g veronal; 7 cc 27% NaOH; 6.5 g Na acetate; 0.19 Na azide make up to I liter with water and XC ml N/I NC1 (pH 8.6). p. 396
394
F. RAPPAPORT,
F. EICHHORN,
J. FISCHL
VOL. 2
0957)
El&ion I. Mark stained areas by pencil lines and cut out in separate segments; then cut each segment into small pieces, and place these in corresponding wide test tubes. 2. Elute in 6 ml 0.02 N NaOH containing 0.2% serum (Reagent 5), shake gently till the paper decolorizes, and let stand for 5 min. 3. Remove the paper pieces with a glass rod. 4. Mix acetic acid (Reagent 4) with formaline (IOO : I) and add I ml of the mixture to each tube, shaking well. 5. Transfer into separate calorimeter tubes and read in Klett-Summerson Electrophotometer-preferably connected to Kipp galvanometer4-using a red filter (600 mp), and setting the zero to reagent mixture. 6. To calculate results, add all calorimeter readings, and, setting the total sum of readings equal to IOO%, obtain relative concentration of fractions.
Fig. 3. Stained electrophoretic strip of serum proteins obtained with the a.m. apparatus mcreased y-globulin).
Example : (’
Fraction
albumin a ,-globulin a,-globulin ,&globulin y-globulin
Fig. 4. Stained electrophoretic strip of serum lipoids, obtained with the a.m. apparatus.
Calorimeter reading
61.20
16 31
4.40 8.60
Z
7.50 18.30
360 Referencesp. 396
%
220
I00.00
VOL. 2 (1957)
APPARATUS FORFILTERPAPERELECTROPHORESIS
395
II. Lifoids Reagents Buffer solution: the same as for proteins. 2. Dye solution : a saturated solution of oil red in boiling, absolute alcohol is prepared ; after cooling, this is diluted with water to 60% alcohol (6 parts dye and 4 parts water), and finally filtered. Filter each time prior to use. The dye can be kept for a long time. 3. 20% v/v acetic acid in ethanol. I.
Procedwe Prepare the apparatus in the same way as for proteins, using Whatman No. I filter paper and a current of 80-90 V. 2. Apply the serum on the starting hne with a pipette, using 0.04 ml for each strip. 3. Let phoresis run for 4 h at room temperature, or for 12-15 h at 5-6°C. 4. Remove strips from apparatus, dry in air, and put them into a drying oven at xoo’C for 20 min. 5. Put one strip into the dye solution overnight. (The second strip is used for cholesterol determination.) 6. Rinse briefly in water to remove color from background and air-dry (Fig. 4). 7 Mark stained a- and /?-fractions areas with a pencil, cut off the segments. Each segment is cut into small pieces, which are placed into corresponding wide test tubes. 8. Extract for I h with 6 ml of 20 ‘$6acetic acid in ethanol, using the same technique as for proteins. 9. Read a- and p-fractions using a green filter (540 m,u), and calculate by the same principle as for proteins. A blank has been prepared from a segment of paper of the same size as the a- and p-fractions and its value accordingly deduced. I.
III.
Cholesterol
Reagents I. Methanol-chloroform mixture (I : 3). 2. Glacial acetic acid. 3. Zlatkis’ color reagents: dissolve IO g ferric chloride in IOO ml acetic acid. Just prior to use, mix one part of this solution with IOO parts of concentrated sulfuric acid.
Compare the strip already preserved (~~~o~~s,Procedwe 5) to its stained twin and cut segments corresponding to the a- and @-fractions. 2. Adjust segments to the same size, taking care that no lipoid-containing paper is lost. A blank of the same size is prepared from the same paper. 3. Cut segments into small fragments, pIace in wide test tubes. Extract cholesterol with 5 ml of methanol-chloroform mixture by IO min shaking, 4. Pour the solvent into 20 x 160 mm test tubes. Repeat washing with 3 ml portions of methanol-chloroform twice, and collect each washing into corresponding test tubes. I.
References p.396
396
F. RAPPAPORT,
F. EICHHORN,
J. FISCHL
VOL. 2
(1957)
Let solvent evaporate at 90°C in an oven. Add 3 ml of glacial acetic acid to each test tube, and mix thoroughly for several min. Add z ml of the color reagent (Reagent 3) and read on the calorimeter, using a green filter and setting the zero to distilled water. Deduct value of reagent blank from both fractions. The sum of the remaining value is treated as IOO~;; the fractions are calculated accordingly. SUMMARY
A detailed description is given of the construction of an inexpensive micro apparatus for filter paper electrophoresis and of methods for the micro-determination of proteins, lipoids, and cholesterol, by electrophoresis, using this apparatus.
Description dCtaillCe de la construction d’un micro-appareil bon march6 pour l’Clectrophor&se B papier filtre et de l’adoption et du dCveloppement de mCt.hodes, pour la micro-dbtermination des protCines, des lipides et du cholest&ol par la phorhse, employant cet appareil. ZUSAMMENFASSUNG
Eine detaillierte Beschreibung des Baus eines billigen Mikro-Apparats fiir PapierElektrophorese wird beschrieben; ebenso such die Einfiihrung und die Entwicklung der Methoden zur Mikrobestimmung von Proteinen, Lipoiden und Cholesterol mit Hilfe dieses Apparates. PE3IOME Aaemx noqm6aoe onmame KOHCTPYK~HII Heaopororo annapa-raw snexqoGope3a na @iAbTposanbnoB6yMare, a TaK)KenpubfeHemieEIpa3smue hieTodoBWKpo-onpeaenenun 6enrcoq mnou.qoB u xonecrepona npki noMoqpi on&icannofoannapala. REFERENCES I G. DISCOMBE, R. F. JONES AND D. P. WINSTANTLEY,J. C&z. Path& 7 (1954) 107. 2 G. T. FRANGLEN, J. Clin. Pathol., 6 (1953) 183. 3 B. ZAK, A. ZLATKIS AND A. J. BOYLE, in E. J. KING, Micro-analysis in Medical Biochemistry, J. &A. Churchill, Ltd., London, 1956, p. 43. 4 F. RAPPAPORT AND F. EICHHORN, Lab. Digest, (1955).
Received May qth,
1957
voL. 2 (1957)
CLINICA CHIMICA ACTA
THE CONSTRUCTION APPARATUS
AND USE OF A SMALL AND INEXPENSIVE
FOR FILTER
F. RAPPAPORT,
Beilinson Hospital.
PAPER
F. EICHHORN
Petah Tikva, and Governmental
ELECTROPHORESIS
AND
J. FISCHL
Hospital. Assaf Harofe, Zrifin (Israel)
Today, filter paper electrophoresis has become an everyday routine work for progressive laboratories. However, the apparatus made by various commercial firms is very expensive and, therefore, not available for laboratories working on a low budget. To overcome financial difficulties, we have constructed an apparatus from cheap and easily obtainable material. In principle, our apparatus is similar to others, with the exception that the electrodes are placed inside and under the paper-supporting bridge, giving the advantage of greatly reduced size over the other type of apparatus. The results obtained by our apparatus are identical to those obtained with the usual types of instruments. Its much smaller size makes electrophoresis in restricted spaces more convenient, ~.g., electrophoresis done under refrigeration, or in small and crowded laboratories. The purpose of this paper is to give a detailed description of the construction of the apparatus and of methods for its use. CONSTRUCTION
OF APPARATUS
The box to be used, made of chloroform-soluble material and obtainable at hardware stores, should have these dimensions: wall thickness 1.2-1.5 mm; length 19.5 cm, width 9.5 cm, and depth 6.0 cm (inner measurements). Four plates (p), 4 x 9.45 cm, are cut out of a sheet of perspex. Two of these plates are inserted perpendicular to the floor and at right angles to the length, each at I cm distance from the midline. The plates, and all additional parts in the apparatus, are glued to the box by perspex material that has been dissolved in chloroform. The 2 cm space in the midsection between the two plates is completely filled with a melted beeswax-paraffin wax (I : I) mixture (w) . (This prevents leakage and short circuit of the system.) Each of the two remaining plates is pierced 20-25 times within a radius of 1.0-1.5 cm from the center with a red-hot needle. These plates are then inserted parallel with the two others, dividing the box into four open compartments (cf. Fig. I). After fixing the plates into their proper place with chloroform-perspex solution, four little squares of IO x 15 mm (s), cut preferably from colored plastic material, are glued to both sides of the plates 7 mm from the upper edge. They serve as marks for the buffer level, and facilitate removal of surplus buffer. Two holes (h), 4 mm in diam., are drilled on one side of the box for the electrodes. They are made at 3.0 cm distance from the center, and at 2.7 cm from the bottom of the box. Two electrodes are made from copper screws about 6 cm long, 4 mm diam., and without head. At one end of each screw a 1.6 cm deep cut is made, and the same side of the screw is filed smooth and someReferences p. 396
VOL.
2 (1957)
APPARATUS FOR FILTER PAPER ELECTROPHORESIS
391
what conical at the end, in order to serve as electrical connector. About 3 mm from the tip of the other end of the screw, a small hole is bored through the metal, and a piece of platinum wire, 4 cm long, is inserted in such a way that the ends of the wire are protruding in a fork-like manner. These ends are then cleaned with HCI, and the wire is welded to the rods with welding alloy. The electrodes are inserted in the box through the holes, with connecting ends protruding 2 cm outside. Two small
19.5
Fig. I. Schematic
representation
of the apparatus.
copper bolts are then screwed on from both sides, fixing the electrodes firmly to the wall of the box. To isolate the bolts, small pieces of perspex plates are glued over them from both sides. To isolate the copper electrode supports from contact with the buffer solution, rubber tubing is used and must cover all the copper surfaces inside the box. Perspex solution in chloroform serves well, both for glueing tubing to metal and for making an isolating film on the copper surface. The rubber tube is pierced at the proper place, and the platinum wire ends are drawn out through the puncture. The end of the tube is closed by a glass plug that is also glued in. To shield the electrodes, two plates of I mm thick perspex material 6.5 x 3.5 cm (c), are bent at 90’ in such a way that one wing is about 2.5 cm and the other 4 cm long. They are glued to the bottom of the box, the longer side running parallel to the compartment-dividing plates, half-way between plate and electrode, the shorter shield wing circling the electrode in the direction of the wax dividing wall. Two small holes of 3 mm diam. (0) are cut into the box walls on each side of the electrode compartment. They are 3.7 cm from the bottom of the box. Rubber tubes of 3 mm length, with a diam. just fitting the holes, are inserted, one end just a few mm inside the box, the other bent upwards, fixed to the outside of the box by a piece of perspex plate. They serve as outlets for the gases generated during electrophoresis. The combined electrode cover-paper support is prepared in the following way : a 2 mm thick perspex plate of the required width (9.45 cm) is inserted into the box, its length being sufficient to cover both electrode compartments (13 cm). When it is inserted, only the two outer compartments are uncovered. This plate is not glued in, References
p. 396
392
F. RAPPAPORT,
F. EICHHORN,
J. FISCHL
VOL. 2
(1957)
but remains a removable part. When inserting it, a piece of polyethylene wrapping material is placed underneath the perspex plate, covering it completely and overlapping upwards 0.5 cm on each side. This ensures air-tightness of the electrode chambers. To prevent the filter paper from sagging during electrophoresis, a device must be made to support the paper strips. Toy racing cars of plastic material will suffice for this. They should be I cm high and z cm long, resting on a firm base. The drivers’
Fig. 2. Photographic
picture of the apparatus.
heads served excellently for supporting the paper strips. Thirty-six of these cars, divided evenly into two sets of three rows each, are glued to the electrode covering plate. Spaces between the rows are about 1.2 cm, and that between the two sets is about 2.2 cm. The paper-holder bridge is made of 4 mm-thick perspex glass. It is 9.4 cm wide, 18 cm long, and 5.5 cm high. The plane on which the paper is supported is 2 cm wide. To ensure good closure of the apparatus, an elastic plastic band (used to insulate electric wires) is glued to the upper edge of the box, carefully connecting the ends with bees’ wax-paraffin mixture. To cover the apparatus a thick (5 mm) glass plate of sufficient size is used. A transformer-rectifier was built in our local electrical workshop. It is equipped with a voltage meter and with a pilot lamp. It has connections for three sets of apparatuses, and is able to supply six to eight more sets if required. No additional apparatus is needed that is not to be found in the usual standard laboratory equipment, e.g., centrifuge, photoelectric calorimeter, pipettes, test tubes, etc. Rejerences fi 396
VOL. 2 (1957)
APPARATUS FOR FILTER PAPER ELECTROPHORESIS
393
METHODS
Reagents I. 2.
3. 4. 5. 6.
9.8 g
sodium barbital, 6.5 g sodium acetate, 0.1 g sodium azide are made up with water to I I; N/I HCl (& IO ml) is added to adapt the pH to 8.6”. 3”/0 sulfosalicylic acid in water. Dye solution: dissolve 0.8 g light green SF1 and 15 g sulfosalicylic acid in 500 ml distilled water. Decolorising solution: acetic acid 2:; soln. in tap zater. Sodium hydroxide X/so, containing 0.27; human serum. Formalin C.P.
Fill apparatus with buffer solution up to the levelling mark in all four compartments, and remove surplus by a water suction pump or with a syringe. with the cover plate, placing polyethylene paper 2. Cover electrode compartments underneath (see above). 3. Fix two strips of Whatman No. r, or Elphor filter paper, 4 x 28 cm, on the paper support by moistening the ends, and stretch tightly. Insert support into apparatus and cover system with cover plate; this should be greased with a thin layer of silicone grease before closing. To ensure airtightness it is advisable to weight down the lid. The whole apparatus should be covered with a cloth to prevent influence of external temperature changes during the electrophoresis. 4. Switch on current of 8o-go V until strips become saturated with buffer, and leave under current for IO more min. 5 For application of the serum on the paper strips, prepare narrow paper ribbons2 from Whatman No. I paper. They should be 2.8 cm x 2 mm, with a 3 mm-folded end on one side. Hold the folded end of one paper ribbon with a plastic forceps, soak it with serum, and place on the corresponding paper strip at the cathode side at about 2 cm from the edge of the paper support. 6. Cover apparatus and leave under current for 15 min. Remove application strips and cover again. 87: Leave under current for 14-16 h. I.
After the strips have been removed from the apparatus, they are air-dried and placed in the sulfosalicylic solution (Reagent 2) for 15 min, in the dye solution for 30 min (Reagent 31, and finally washed with reagent No. 4 until the paper background is completely decolorised. They are then air-dried (see Fig. 3). * When
exactly (NaN,);
Referestces
no sodium barbital C.P. is available, the following buffer may be used, which gives the same results: 8.8. g veronal; 7 cc 27% NaOH; 6.5 g Na acetate; 0.19 Na azide make up to I liter with water and XC ml N/I NC1 (pH 8.6). p. 396
394
F. RAPPAPORT,
F. EICHHORN,
J. FISCHL
VOL. 2
0957)
El&ion I. Mark stained areas by pencil lines and cut out in separate segments; then cut each segment into small pieces, and place these in corresponding wide test tubes. 2. Elute in 6 ml 0.02 N NaOH containing 0.2% serum (Reagent 5), shake gently till the paper decolorizes, and let stand for 5 min. 3. Remove the paper pieces with a glass rod. 4. Mix acetic acid (Reagent 4) with formaline (IOO : I) and add I ml of the mixture to each tube, shaking well. 5. Transfer into separate calorimeter tubes and read in Klett-Summerson Electrophotometer-preferably connected to Kipp galvanometer4-using a red filter (600 mp), and setting the zero to reagent mixture. 6. To calculate results, add all calorimeter readings, and, setting the total sum of readings equal to IOO%, obtain relative concentration of fractions.
Fig. 3. Stained electrophoretic strip of serum proteins obtained with the a.m. apparatus mcreased y-globulin).
Example : (’
Fraction
albumin a ,-globulin a,-globulin ,&globulin y-globulin
Fig. 4. Stained electrophoretic strip of serum lipoids, obtained with the a.m. apparatus.
Calorimeter reading
61.20
16 31
4.40 8.60
Z
7.50 18.30
360 Referencesp. 396
%
220
I00.00
VOL. 2 (1957)
APPARATUS FORFILTERPAPERELECTROPHORESIS
395
II. Lifoids Reagents Buffer solution: the same as for proteins. 2. Dye solution : a saturated solution of oil red in boiling, absolute alcohol is prepared ; after cooling, this is diluted with water to 60% alcohol (6 parts dye and 4 parts water), and finally filtered. Filter each time prior to use. The dye can be kept for a long time. 3. 20% v/v acetic acid in ethanol. I.
Procedwe Prepare the apparatus in the same way as for proteins, using Whatman No. I filter paper and a current of 80-90 V. 2. Apply the serum on the starting hne with a pipette, using 0.04 ml for each strip. 3. Let phoresis run for 4 h at room temperature, or for 12-15 h at 5-6°C. 4. Remove strips from apparatus, dry in air, and put them into a drying oven at xoo’C for 20 min. 5. Put one strip into the dye solution overnight. (The second strip is used for cholesterol determination.) 6. Rinse briefly in water to remove color from background and air-dry (Fig. 4). 7 Mark stained a- and /?-fractions areas with a pencil, cut off the segments. Each segment is cut into small pieces, which are placed into corresponding wide test tubes. 8. Extract for I h with 6 ml of 20 ‘$6acetic acid in ethanol, using the same technique as for proteins. 9. Read a- and p-fractions using a green filter (540 m,u), and calculate by the same principle as for proteins. A blank has been prepared from a segment of paper of the same size as the a- and p-fractions and its value accordingly deduced. I.
III.
Cholesterol
Reagents I. Methanol-chloroform mixture (I : 3). 2. Glacial acetic acid. 3. Zlatkis’ color reagents: dissolve IO g ferric chloride in IOO ml acetic acid. Just prior to use, mix one part of this solution with IOO parts of concentrated sulfuric acid.
Compare the strip already preserved (~~~o~~s,Procedwe 5) to its stained twin and cut segments corresponding to the a- and @-fractions. 2. Adjust segments to the same size, taking care that no lipoid-containing paper is lost. A blank of the same size is prepared from the same paper. 3. Cut segments into small fragments, pIace in wide test tubes. Extract cholesterol with 5 ml of methanol-chloroform mixture by IO min shaking, 4. Pour the solvent into 20 x 160 mm test tubes. Repeat washing with 3 ml portions of methanol-chloroform twice, and collect each washing into corresponding test tubes. I.
References p.396
396
F. RAPPAPORT,
F. EICHHORN,
J. FISCHL
VOL. 2
(1957)
Let solvent evaporate at 90°C in an oven. Add 3 ml of glacial acetic acid to each test tube, and mix thoroughly for several min. Add z ml of the color reagent (Reagent 3) and read on the calorimeter, using a green filter and setting the zero to distilled water. Deduct value of reagent blank from both fractions. The sum of the remaining value is treated as IOO~;; the fractions are calculated accordingly. SUMMARY
A detailed description is given of the construction of an inexpensive micro apparatus for filter paper electrophoresis and of methods for the micro-determination of proteins, lipoids, and cholesterol, by electrophoresis, using this apparatus.
Description dCtaillCe de la construction d’un micro-appareil bon march6 pour l’Clectrophor&se B papier filtre et de l’adoption et du dCveloppement de mCt.hodes, pour la micro-dbtermination des protCines, des lipides et du cholest&ol par la phorhse, employant cet appareil. ZUSAMMENFASSUNG
Eine detaillierte Beschreibung des Baus eines billigen Mikro-Apparats fiir PapierElektrophorese wird beschrieben; ebenso such die Einfiihrung und die Entwicklung der Methoden zur Mikrobestimmung von Proteinen, Lipoiden und Cholesterol mit Hilfe dieses Apparates. PE3IOME Aaemx noqm6aoe onmame KOHCTPYK~HII Heaopororo annapa-raw snexqoGope3a na @iAbTposanbnoB6yMare, a TaK)KenpubfeHemieEIpa3smue hieTodoBWKpo-onpeaenenun 6enrcoq mnou.qoB u xonecrepona npki noMoqpi on&icannofoannapala. REFERENCES I G. DISCOMBE, R. F. JONES AND D. P. WINSTANTLEY,J. C&z. Path& 7 (1954) 107. 2 G. T. FRANGLEN, J. Clin. Pathol., 6 (1953) 183. 3 B. ZAK, A. ZLATKIS AND A. J. BOYLE, in E. J. KING, Micro-analysis in Medical Biochemistry, J. &A. Churchill, Ltd., London, 1956, p. 43. 4 F. RAPPAPORT AND F. EICHHORN, Lab. Digest, (1955).
Received May qth,
1957