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A method of preparing agarose
BIOCHIMICA ET BIOPHYSICA ACTA
169
BBA 25 024
A METHOD OF PREPARING
AGAROSE
]3. RUSSELL, T. H. MEAD AND A. POLSON
The C.S.I.R. and U.C.T. Virus Research Unit, Department of Pathology, University of Cape Town, Cape Town (South Africa) (Received August 6th, 1963)
SUMMARY
I. Agarose has been prepared b y fractional precipitation of agar with polyethylene glycol. 2. Although relatively simple, the method ensures a product essentially free from sulphate and possessing a minimum of eleetroendosmotic and ion-exchange properties. 3. Applications to tissue culture and physico-chemical methods are described.
INTRODUCTION
ARAKI1 has shown that agar consists of two fractions, a neutral galactose polymer (agarose) and a sulphated polysaccharide (agaropectin) which imparts to the agarose properties undesirable for certain applications. Agarose has been prepared b y the acetylation method of ARAKI1 and the cetylpyridinium chloride method of HJERTt~N ~. The acetylation method is very tedious and expensive. The final product as prepared b y us had a lower gel strength than the original agar and still retained some negatively charged impurities. The cetylpyridinium chloride method appears to effect an excellent separation of the two components but is expensive. The method to be described depends on fractional precipitation with polyethylene glycol and avoids the use of expensive chemicals. MATERIALS AND METHODS
Agar. Ionagar No. 2 (Oxo Ltd. London, EC4) has been used for most of this work but satisfactory results have been obtained with agar from other sources. Polyethylene glycol (tool. wt. 6000) was obtained from Shell Chemicals.
Estimation of sulphate Sulphate was estimated b y the method of DODGSON AND PRICEs modified for use with a turbidimeter. Leaf gelatin (2 g) was dissolved in water (400 ml) at 60o-7 °0 and the solution, after standing about 48 h at 4 ° was treated at room temperature with barium chloride dihydrate (2 g). After a night at 4 ° the cloudy solution was centrifuged for I h at Ioooo rev./min in the Spinco No. 30 rotor at room temperature. The clear supernatant, treated with thiomersal (o.oi %) appeared stable for Abbreviation: p.e.g.,
polyethylene glycol. Biochim. Biophys. Acta, 86 (1964) 169-174
i70
B. R U S S E L L ,
T. H. 3{EAD, A. P O L S O X
m a n y weeks. Standards were prepared containing in o.2 ml Io, 2o~ 30, 4o, 50, 6o, 7o, 8o, 9 ° and Ioo/~g sulphate. Test mixtures containing 0.2 ml standard (or o.2 ml He{) for the blank), 0.2 ml I N HC1, 0.3 ml I N sodium acetate, 3.3 ml H20 and z m! of the gelatin-BaCls solution were examined in the turbidimeter at known time intervals after preparation. The turbidimeter was the light-scattering apparatus of OsTEX~' modified as described b y M E A n 5 and consisting essentially of a square perspex eel! illuminated b y a narrow- pencil of light. A photomultiplier cell at right angles to the incident beam measured light scattered b y a n y particles in the eel1 contents, l"or these experiments the galvanometer was set to zero with the bIank in the cell a!)A to ~oo divisions with the standard containing xoo b*g sulphate. Readings were taken about 2o rain after addition of the gelatin BaCI 2 solution and usually repeated (after the samples had been shaken) on the following day with similar results. Galvanometer readings plotted against /,g sulphate gave a straight line in the 4o-9o-fzg range. The agar and agarose samples (about zo mg) were hydrolysed in sealed tubes with I.o-ml amounts of I N HC1 at zo5° for 5 h. The tubes were centrifuged briefly to compact the traces of humin before being opened. I n a preliminary experiment o.2 ml hydrolysate and o.z m] HzO replaced the s t a n d a r d and the I N HC1 in the test mixture. I n subsequent assays of each hydrolysate, mixtures of o.I ml or o.z m! hydrolysate + 2 ml of one or more of the standards were set up in a series which also included the standards in the 4o-9o-/,g range. In this w a y the turbidity of the sample was compared with t h a t of the standards at several levets in the range in which ~g sulphate and galvonometer readings were linearly related. Electroe~dosmosis measurements Method A : R a b b i t serum was submitted to electrophoresis in the agar or agarose sample at p H 8.2 under s t a n d a r d conditions and the direction and extent of migration of the y-globulin and proteins was taken as a measure of electroendosmotic flow. The electrophoresis was carried out on microscope slides resting on the water-cooled (Io °) floor of an enclosed perspex apparatus. Strips of filter paper carried the current between the electrode vessels and the slides. Buffer prepared according to GRABAR AM) WILLIamS~ b y mixing a o.I M solution of sodium diethylbarbiturate (77o ml) with o.I N HC1 (23o mi) was used in the electrode vessels and paper strips and buffer of half this concentration for preparing the slides which carried 2.5 ml of x % solution of the agar to be tested. The serum was placed in a hole about I.o m m in diameter at the mid point of the slide and electrophoresis was continued for I h at ~o V/cm. The slides were immersed for xo min in a fixing solution containing in 50o ml : HgCI> 2 g; acetic acid, I o ml and ethanol, 30o ml. T h e y were stained with naphthalene black or nigrosin. Method B This was carried out in the same w a y except t h a t a I T (w/v) aqueous solution of p.e.g, was used in place of rabbit serum. After electrophoresis the slide was immersed in I5 % trichloroacetic acid for about Io rain. This revealed the final position of the glycol b y the formation of a white spot. EXPERIMENTAL
Agar, even from one supplier, is so variable t h a t preliminary tests should be made on each new batch to find the lowest concentration of p.e.g, giving reasonably BCoc/~Cm. B{o~]~ys. ~c~a, 86 (i964) 169 !74
A METHOD OF PREPARING AGAROSE
I7I
complete precipitation of the agarose fraction. The precipitates obtained at varied concentrations of p.e.g, are tested b y Methods A or B above. We have found values between 7 % and 2o %. The description which follows applies to a batch requiring 20 % (w/v),
Preparation of agarose Agar (80 g) is stirred mechanically with distilled water (2 1) on a boiling-water bath until completely dissolved. To the solution at 8o ° are added 2 1 of 4 ° !/o (w/v) p.e.g, also at 8o ° and the whole is mixed well. Within 3-5 min a flocculent precipitate settles to the bottom of the containing vessel. The precipitate is separated from the supernatant fluid b y filtration through I I o - m e s h nylon cloth and is immediately placed, still inside the cloth, in a basin containing water at 4 o°. Most of the agaropectin remains in the mother liquor and more is removed by washing at 4 o°. It is, however, important not to wash at 4 °0 for more than 2-3 min as the precipitate itself is soluble at this temperature. Alternatively the agarose precipitate m a y be collected and washed in a prewarmed basket centrifuge lined with nylon cloth. The nylon bag containing the precipitate is then transferred to water at about i5 ° in which it is thoroughly washed and the larger pieces broken up. Most of tile remaining p.e.g, is removed b y stirring the precipitate in 5 1 of distilled water overnight. The washed granules are collected in i I o - m e s h nylon cloth either in a basket centrifuge or Buchner funnel, washed with acetone and dried in a current of warm air. The product is weighed, dissolved in distilled water at Ioo ° to form a 4 % (w/v) solution, reprecipitated with p.e:g, and washed and dried in the same way. A third precipitation is usually necessary to ensure complete removal of agaropectin. Sometimes, especially at the second and third precipitation stages, enough sodium chloride to give a final concentration of o.5 % must be added after the p.e.g, to flocculate the agarose in a form suitable for collection. The final product is stirred overnight with distilled water (5 1), collected and washed with water on a nylon cloth in a basket centrifuge, ground in a mortar, stirred again overnight with water, centrifuged, washed with acetone and dried in warm air. No p.e.g, should be detectable by the addition to a few drops of the final agarose washings of an equal volume of a saturated solution of trichloroacetic acid. A yield of 6o-7o % of partially purified agar is obtained after the first precipitation. The final yield of agarose varied between 30 and 45 %The quantities indicated above have been found satisfactory for one batch of Ionagar No. 2 but it must be stressed that these will vary for agars derived from different sources and even for different batches of the same product. Thus it has been found that 7 % (w/v) of p.e.g, was sufficient to precipitate agarose from agar derived from Gdidium pristoides. The product thus obtained was found suitable for electrophoresis, gel diffusionfiltration and as overlay for virus plaque formation. As, however, commercial agar seldom forms completely clear aqueous solutions we have on occasion clarified initial I or 2 % solutions before addition of p.e.g, by passing them at about 5 1/h through the clarifying bowl of a Model I A.P. Sharples centrifuge driven b y steam turbine at 40000-50000 rev./min. The machine was heated b y passing steam through the ~coils normally used for refrigeration and the fluid entered through a small Liebig condenser with steam in the jacket.
Biochim. Biophys. Acts, 86 (1964) 1 6 9 - i 7 4
172
B. RUSSELL, T° H. MEAD~ A. POLSON
Pfeparatiolz of ~garopecti~ The agaropectin may be separated from the supernatant fluid of the first precipitate. The solution is cooled to 5 ° and mixed with an equal volume of acetore A fine white precipitate of agaropectin is formed which settles to the bottom of the containing vessel when the mixture is heated in a water bath at 5 °° for 5 rain. The mother liquor is decanted and the agaropectin shaken with chloroform in a separating funnel to remove most of the p.e.g, and then washed with water by stirring untii the trichloroacetic acid test for p.e.g, is negative. The product is dehydrated with acetone and dried in warm air. RESULTS
Each batch of agarose was tested for electroendosmotic effect as described under M~THODS and some have been ana!ysed for sulphate. In agreement with HJERT~2N9 we have invariably found some electroendosmosis even in samples which have been precipitated 5 times with p.e,g. Fig. x shows the results of electrophoresis under standard conditions of rabbit serum in ~ % gels of; a, agarose; b, gel obtained from the supernatant fluid after removal of the agarose; c, unfractionated tonagar No. 2; d, purified agaropectin. Eiectroendosmosis in the Ionagar was, as expected, intermediate between that in the agarose and agaropectln fractions. In Fig. 2 electroendosmotic flow in agarose and in !onagar are compared by Method B using p.e.g, as indicator. Sulphate contents of o.75 %, o.78 % and I.Z % were found in Ionagar on different
Fig. I. ]Electrophoresis of rabbit serul~l in diflerent agar fractions, a, agarose; b, partially purified agaropectin; c, unfractionated lonagar and d, purified agaropectin. In all cases the sequence of components in the diagrams from the top is albumin, a-globulins, fl-globulin and T-globuli1~. ~Note the large e]ectroendosmosis in diagram d.
Biochim. Biophys. Act~, 86 (I964) t69 i 7 4
A METHOD OF PREPARING AGAROSE
173
occasions but no sulphate was detectable by the method used in two batches of agarose. The possibility of a trace of sulphate being adsorbed to the humin produced on hydrolysis cannot, however, be excluded.
Fig. 2. Electroendosmotic effect as indicated b y polyethylene glycol; p.e.g, was introduced ill the centre hole. I n a, the s u p p o r t i n g m e d i u m was agarose a n d in b, I o n a g a r . DISCUSSION
For electrophoresis and immunoelectrophoresis the advantages of agarose as compared with agar have been pointed out by HJERT]~N9. Agarose prepared by the p.e.g. method has been found very suitable for these applications. For gel diffusion--filtration which is usually intended to fraetionate mixtures solely on a diffusibility basis, the negatively charged agaropectin can introduce complications especially at low ionic strengths. Haemoglobin, for example, in 0.005 M phosphate is held tenaciously by crude unfractionated agar gels of a concentration allowing rapid filtration of other proteins comparable in diffusion rate. Although advantage has been taken in some instances (PoLsON AND LEVITT, to be published) to secure better fractionation of protein mixtures by this ion-exchange effect than could be obtained with agarose at higher ionic strengths, it is clear that agarose is, generally preferable to agar for gel diffusion-filtration1° especially if conclusions about diffusibility are to be drawn from the results. Another advantage of agarose for this purpose is that its granulated gels are less easily deformed than those prepared from agar and columns of granulated agarose have proved capable of retaining their fractionating properties unchanged during storage under buffer at room temperatures for m a n y months. Application of the agar overlay plaque counting method of titration and clone selection to certain viruses is complicated b y the presence in agar of a substance Biochim. Biophys. Acta, 86 (1964) 169-174-
i74
B. RUSSELL, T. H. MEAD, A. POLSON
h a v i n g a m a r k e d effect o n p l a q u e size a n d n u m b e r s . TAKE>IOTO AND LIE>I-IABER:have s h o w n t h a t t h i s is d u e t o a s u l p h a t e d p o l y s a c e h a r i d e . A g a r o s e p r e p a r e d b y t h e p.e.g. m e t h o d s e e m s t o b e free of t h i s v i r u s i n h i b i t o r as it h a s p r o v e d v e r y s u i t a b l e f,~}r u s e in t h e o v e r l a y t e c h n i q u e ~ ACKNOWLEDGEMENTS T h e a u t h o r s are i n d e b t e d t o P r o f e s s o r A. K I P P s for his c o n t i n u e d i n t e r e s t in t h i s w o r k °
RE~ERENCES 1 C..A..RAKI,J. Chem. Soc. JaVa7% 19~re Chem. Sec[., 58 (r937) I338. 2 S . IcIJERTklV, Biochim. Biophys. AcLa, 62 (I962) 445a K. S. DODOSON A~D R. G. PRICE, Biochem. J., 84 (1962) lO6. 4 G. OSTER, J. Gen. Physiol., 33 (I95o) 445. 5 T. H. MEAD, J. Ge•. Micvobiol., 27 (1962) 397. 6 p. GRABaR A~D C. A. V~TILLIa~S, Biochim. Biophys. Acta, 17 (1955) 67. 7 K. N. TAKEMOTO AND H. LIEBHABER, ]/irO[Ogy, 14 (1961) 456. S V. I. AGOL AND M. YA CHUMAKOVA,VCrology, I 7 (1962) 22!. 9 S. HJERTkN, Biochim. Biophys. AcXa, 53 (I96~) 514 • lO S. HJERT~N, Af6h. Biochem. Biophys,, 99 (1962) 466.
Bioc/dm. Biophys. ActcC 86 (x964) 169-!74
169
BBA 25 024
A METHOD OF PREPARING
AGAROSE
]3. RUSSELL, T. H. MEAD AND A. POLSON
The C.S.I.R. and U.C.T. Virus Research Unit, Department of Pathology, University of Cape Town, Cape Town (South Africa) (Received August 6th, 1963)
SUMMARY
I. Agarose has been prepared b y fractional precipitation of agar with polyethylene glycol. 2. Although relatively simple, the method ensures a product essentially free from sulphate and possessing a minimum of eleetroendosmotic and ion-exchange properties. 3. Applications to tissue culture and physico-chemical methods are described.
INTRODUCTION
ARAKI1 has shown that agar consists of two fractions, a neutral galactose polymer (agarose) and a sulphated polysaccharide (agaropectin) which imparts to the agarose properties undesirable for certain applications. Agarose has been prepared b y the acetylation method of ARAKI1 and the cetylpyridinium chloride method of HJERTt~N ~. The acetylation method is very tedious and expensive. The final product as prepared b y us had a lower gel strength than the original agar and still retained some negatively charged impurities. The cetylpyridinium chloride method appears to effect an excellent separation of the two components but is expensive. The method to be described depends on fractional precipitation with polyethylene glycol and avoids the use of expensive chemicals. MATERIALS AND METHODS
Agar. Ionagar No. 2 (Oxo Ltd. London, EC4) has been used for most of this work but satisfactory results have been obtained with agar from other sources. Polyethylene glycol (tool. wt. 6000) was obtained from Shell Chemicals.
Estimation of sulphate Sulphate was estimated b y the method of DODGSON AND PRICEs modified for use with a turbidimeter. Leaf gelatin (2 g) was dissolved in water (400 ml) at 60o-7 °0 and the solution, after standing about 48 h at 4 ° was treated at room temperature with barium chloride dihydrate (2 g). After a night at 4 ° the cloudy solution was centrifuged for I h at Ioooo rev./min in the Spinco No. 30 rotor at room temperature. The clear supernatant, treated with thiomersal (o.oi %) appeared stable for Abbreviation: p.e.g.,
polyethylene glycol. Biochim. Biophys. Acta, 86 (1964) 169-174
i70
B. R U S S E L L ,
T. H. 3{EAD, A. P O L S O X
m a n y weeks. Standards were prepared containing in o.2 ml Io, 2o~ 30, 4o, 50, 6o, 7o, 8o, 9 ° and Ioo/~g sulphate. Test mixtures containing 0.2 ml standard (or o.2 ml He{) for the blank), 0.2 ml I N HC1, 0.3 ml I N sodium acetate, 3.3 ml H20 and z m! of the gelatin-BaCls solution were examined in the turbidimeter at known time intervals after preparation. The turbidimeter was the light-scattering apparatus of OsTEX~' modified as described b y M E A n 5 and consisting essentially of a square perspex eel! illuminated b y a narrow- pencil of light. A photomultiplier cell at right angles to the incident beam measured light scattered b y a n y particles in the eel1 contents, l"or these experiments the galvanometer was set to zero with the bIank in the cell a!)A to ~oo divisions with the standard containing xoo b*g sulphate. Readings were taken about 2o rain after addition of the gelatin BaCI 2 solution and usually repeated (after the samples had been shaken) on the following day with similar results. Galvanometer readings plotted against /,g sulphate gave a straight line in the 4o-9o-fzg range. The agar and agarose samples (about zo mg) were hydrolysed in sealed tubes with I.o-ml amounts of I N HC1 at zo5° for 5 h. The tubes were centrifuged briefly to compact the traces of humin before being opened. I n a preliminary experiment o.2 ml hydrolysate and o.z m] HzO replaced the s t a n d a r d and the I N HC1 in the test mixture. I n subsequent assays of each hydrolysate, mixtures of o.I ml or o.z m! hydrolysate + 2 ml of one or more of the standards were set up in a series which also included the standards in the 4o-9o-/,g range. In this w a y the turbidity of the sample was compared with t h a t of the standards at several levets in the range in which ~g sulphate and galvonometer readings were linearly related. Electroe~dosmosis measurements Method A : R a b b i t serum was submitted to electrophoresis in the agar or agarose sample at p H 8.2 under s t a n d a r d conditions and the direction and extent of migration of the y-globulin and proteins was taken as a measure of electroendosmotic flow. The electrophoresis was carried out on microscope slides resting on the water-cooled (Io °) floor of an enclosed perspex apparatus. Strips of filter paper carried the current between the electrode vessels and the slides. Buffer prepared according to GRABAR AM) WILLIamS~ b y mixing a o.I M solution of sodium diethylbarbiturate (77o ml) with o.I N HC1 (23o mi) was used in the electrode vessels and paper strips and buffer of half this concentration for preparing the slides which carried 2.5 ml of x % solution of the agar to be tested. The serum was placed in a hole about I.o m m in diameter at the mid point of the slide and electrophoresis was continued for I h at ~o V/cm. The slides were immersed for xo min in a fixing solution containing in 50o ml : HgCI> 2 g; acetic acid, I o ml and ethanol, 30o ml. T h e y were stained with naphthalene black or nigrosin. Method B This was carried out in the same w a y except t h a t a I T (w/v) aqueous solution of p.e.g, was used in place of rabbit serum. After electrophoresis the slide was immersed in I5 % trichloroacetic acid for about Io rain. This revealed the final position of the glycol b y the formation of a white spot. EXPERIMENTAL
Agar, even from one supplier, is so variable t h a t preliminary tests should be made on each new batch to find the lowest concentration of p.e.g, giving reasonably BCoc/~Cm. B{o~]~ys. ~c~a, 86 (i964) 169 !74
A METHOD OF PREPARING AGAROSE
I7I
complete precipitation of the agarose fraction. The precipitates obtained at varied concentrations of p.e.g, are tested b y Methods A or B above. We have found values between 7 % and 2o %. The description which follows applies to a batch requiring 20 % (w/v),
Preparation of agarose Agar (80 g) is stirred mechanically with distilled water (2 1) on a boiling-water bath until completely dissolved. To the solution at 8o ° are added 2 1 of 4 ° !/o (w/v) p.e.g, also at 8o ° and the whole is mixed well. Within 3-5 min a flocculent precipitate settles to the bottom of the containing vessel. The precipitate is separated from the supernatant fluid b y filtration through I I o - m e s h nylon cloth and is immediately placed, still inside the cloth, in a basin containing water at 4 o°. Most of the agaropectin remains in the mother liquor and more is removed by washing at 4 o°. It is, however, important not to wash at 4 °0 for more than 2-3 min as the precipitate itself is soluble at this temperature. Alternatively the agarose precipitate m a y be collected and washed in a prewarmed basket centrifuge lined with nylon cloth. The nylon bag containing the precipitate is then transferred to water at about i5 ° in which it is thoroughly washed and the larger pieces broken up. Most of tile remaining p.e.g, is removed b y stirring the precipitate in 5 1 of distilled water overnight. The washed granules are collected in i I o - m e s h nylon cloth either in a basket centrifuge or Buchner funnel, washed with acetone and dried in a current of warm air. The product is weighed, dissolved in distilled water at Ioo ° to form a 4 % (w/v) solution, reprecipitated with p.e:g, and washed and dried in the same way. A third precipitation is usually necessary to ensure complete removal of agaropectin. Sometimes, especially at the second and third precipitation stages, enough sodium chloride to give a final concentration of o.5 % must be added after the p.e.g, to flocculate the agarose in a form suitable for collection. The final product is stirred overnight with distilled water (5 1), collected and washed with water on a nylon cloth in a basket centrifuge, ground in a mortar, stirred again overnight with water, centrifuged, washed with acetone and dried in warm air. No p.e.g, should be detectable by the addition to a few drops of the final agarose washings of an equal volume of a saturated solution of trichloroacetic acid. A yield of 6o-7o % of partially purified agar is obtained after the first precipitation. The final yield of agarose varied between 30 and 45 %The quantities indicated above have been found satisfactory for one batch of Ionagar No. 2 but it must be stressed that these will vary for agars derived from different sources and even for different batches of the same product. Thus it has been found that 7 % (w/v) of p.e.g, was sufficient to precipitate agarose from agar derived from Gdidium pristoides. The product thus obtained was found suitable for electrophoresis, gel diffusionfiltration and as overlay for virus plaque formation. As, however, commercial agar seldom forms completely clear aqueous solutions we have on occasion clarified initial I or 2 % solutions before addition of p.e.g, by passing them at about 5 1/h through the clarifying bowl of a Model I A.P. Sharples centrifuge driven b y steam turbine at 40000-50000 rev./min. The machine was heated b y passing steam through the ~coils normally used for refrigeration and the fluid entered through a small Liebig condenser with steam in the jacket.
Biochim. Biophys. Acts, 86 (1964) 1 6 9 - i 7 4
172
B. RUSSELL, T° H. MEAD~ A. POLSON
Pfeparatiolz of ~garopecti~ The agaropectin may be separated from the supernatant fluid of the first precipitate. The solution is cooled to 5 ° and mixed with an equal volume of acetore A fine white precipitate of agaropectin is formed which settles to the bottom of the containing vessel when the mixture is heated in a water bath at 5 °° for 5 rain. The mother liquor is decanted and the agaropectin shaken with chloroform in a separating funnel to remove most of the p.e.g, and then washed with water by stirring untii the trichloroacetic acid test for p.e.g, is negative. The product is dehydrated with acetone and dried in warm air. RESULTS
Each batch of agarose was tested for electroendosmotic effect as described under M~THODS and some have been ana!ysed for sulphate. In agreement with HJERT~2N9 we have invariably found some electroendosmosis even in samples which have been precipitated 5 times with p.e,g. Fig. x shows the results of electrophoresis under standard conditions of rabbit serum in ~ % gels of; a, agarose; b, gel obtained from the supernatant fluid after removal of the agarose; c, unfractionated tonagar No. 2; d, purified agaropectin. Eiectroendosmosis in the Ionagar was, as expected, intermediate between that in the agarose and agaropectln fractions. In Fig. 2 electroendosmotic flow in agarose and in !onagar are compared by Method B using p.e.g, as indicator. Sulphate contents of o.75 %, o.78 % and I.Z % were found in Ionagar on different
Fig. I. ]Electrophoresis of rabbit serul~l in diflerent agar fractions, a, agarose; b, partially purified agaropectin; c, unfractionated lonagar and d, purified agaropectin. In all cases the sequence of components in the diagrams from the top is albumin, a-globulins, fl-globulin and T-globuli1~. ~Note the large e]ectroendosmosis in diagram d.
Biochim. Biophys. Act~, 86 (I964) t69 i 7 4
A METHOD OF PREPARING AGAROSE
173
occasions but no sulphate was detectable by the method used in two batches of agarose. The possibility of a trace of sulphate being adsorbed to the humin produced on hydrolysis cannot, however, be excluded.
Fig. 2. Electroendosmotic effect as indicated b y polyethylene glycol; p.e.g, was introduced ill the centre hole. I n a, the s u p p o r t i n g m e d i u m was agarose a n d in b, I o n a g a r . DISCUSSION
For electrophoresis and immunoelectrophoresis the advantages of agarose as compared with agar have been pointed out by HJERT]~N9. Agarose prepared by the p.e.g. method has been found very suitable for these applications. For gel diffusion--filtration which is usually intended to fraetionate mixtures solely on a diffusibility basis, the negatively charged agaropectin can introduce complications especially at low ionic strengths. Haemoglobin, for example, in 0.005 M phosphate is held tenaciously by crude unfractionated agar gels of a concentration allowing rapid filtration of other proteins comparable in diffusion rate. Although advantage has been taken in some instances (PoLsON AND LEVITT, to be published) to secure better fractionation of protein mixtures by this ion-exchange effect than could be obtained with agarose at higher ionic strengths, it is clear that agarose is, generally preferable to agar for gel diffusion-filtration1° especially if conclusions about diffusibility are to be drawn from the results. Another advantage of agarose for this purpose is that its granulated gels are less easily deformed than those prepared from agar and columns of granulated agarose have proved capable of retaining their fractionating properties unchanged during storage under buffer at room temperatures for m a n y months. Application of the agar overlay plaque counting method of titration and clone selection to certain viruses is complicated b y the presence in agar of a substance Biochim. Biophys. Acta, 86 (1964) 169-174-
i74
B. RUSSELL, T. H. MEAD, A. POLSON
h a v i n g a m a r k e d effect o n p l a q u e size a n d n u m b e r s . TAKE>IOTO AND LIE>I-IABER:have s h o w n t h a t t h i s is d u e t o a s u l p h a t e d p o l y s a c e h a r i d e . A g a r o s e p r e p a r e d b y t h e p.e.g. m e t h o d s e e m s t o b e free of t h i s v i r u s i n h i b i t o r as it h a s p r o v e d v e r y s u i t a b l e f,~}r u s e in t h e o v e r l a y t e c h n i q u e ~ ACKNOWLEDGEMENTS T h e a u t h o r s are i n d e b t e d t o P r o f e s s o r A. K I P P s for his c o n t i n u e d i n t e r e s t in t h i s w o r k °
RE~ERENCES 1 C..A..RAKI,J. Chem. Soc. JaVa7% 19~re Chem. Sec[., 58 (r937) I338. 2 S . IcIJERTklV, Biochim. Biophys. AcLa, 62 (I962) 445a K. S. DODOSON A~D R. G. PRICE, Biochem. J., 84 (1962) lO6. 4 G. OSTER, J. Gen. Physiol., 33 (I95o) 445. 5 T. H. MEAD, J. Ge•. Micvobiol., 27 (1962) 397. 6 p. GRABaR A~D C. A. V~TILLIa~S, Biochim. Biophys. Acta, 17 (1955) 67. 7 K. N. TAKEMOTO AND H. LIEBHABER, ]/irO[Ogy, 14 (1961) 456. S V. I. AGOL AND M. YA CHUMAKOVA,VCrology, I 7 (1962) 22!. 9 S. HJERTkN, Biochim. Biophys. AcXa, 53 (I96~) 514 • lO S. HJERT~N, Af6h. Biochem. Biophys,, 99 (1962) 466.
Bioc/dm. Biophys. ActcC 86 (x964) 169-!74