183

Clinica Chimica Acta, 85 (1978) 183-191 0 Elsevier/North-Holland Biomedical Press

CGA 9304

ISOELECTRIC FOCUSSING GEL MEMBRANES

OF PROTEINS

ON CELLULOSE

ACETATE

JEFFREY AMBLER Department

of Clinical Chemistry, General Hospital, Nottingham,

NGl 6HA (U.K.)

(Received November 4th, 1977)

Summary A method is described for analytical isoelectric focussing of proteins on cellulose acetate gel strips pretreated with boron trifluoride in methanol. It is satisfactory for separations requiring narrow pH gradients as well as for those requiring wide gradients. As well as conventional protein staining methods, the use of immunological reagents is described to identify directly specific proteins in a complex separation. The method is extremely quick and easy to perform and requires only low voltage electrophoresis equipment.

Introduction Separation of charged species by analytical isoelectric focussing is usually carried out in polyacrylamide gel rods [l] or slabs [2]. Although the technique has great potential value in clinical laboratories, its use has been restricted, partly because the preparation of ampholyte containing gels is tedious and difficult and partly because the equipment, including a high voltage power pack, is often not available in a routine situation. Although cellulose acetate type materials are widely used for electrophoresis, they do not appear to be suitable for isoelectric focussing because of interaction between carrier ampholytes and charged groups in the membrane. This paper describes how cellulose acetate gel strips (Cellogel@) may be modified by treatment with boron trifluoride in methanol so as to become a suitable support material for low voltage isoelectric focussing. Apart from the ease of working with a solid material, the use of cellulose acetate gel strips also revolutionises protein detection following the focussing separation. Hence direct immunofixation of proteins using specific antisera is possible and is also described in this paper. Immunofocussing adds another dimension to the separation of proteins.

Mate~als and methods 1. Apparatus

The electrophoresis tank (Model U 77) and the power pack (Vokam SAE 2761) were manufactured by Shandon - Southern. A perspex water cooled platen with anticondensation lid was also used. The tank must be closed to the atmosphere and the power pack must be capable of delivering a constant 500 volts. 2. Pretreatment

of the cel2uloseacetate

gel strips

Cellogel strips (5.7 X 14 cm) were obtained from Whatman Labsales Ltd. They were pretreated by methylation to remove the free carboxyf groups. This was effected by transferring 25 strips through 2 X 100 ml methanol (AR) washes and into 150 ml boron trifluoride (5.0%) in methanol (prepared from 14% boron trifluoride in methanol B.D.H. Ltd.). The strips were shaken with the methylation reagent for 10 min at room temperature to ensure even penetration before being incubated at 45°C for 50 min. After a further lo-mm shaking at room temperature the modified Cellogel strips were washed with 3 X 100 ml of methanol (AR) to remove excess boron trifluoride reagent and they were stored in this solvent until required. 3. Introduction of am~ho~~tes Ampholytes (Ampholine, LKB Ltd.) were obtained as 40% solutions in various pH ranges. Gradients of choice were constructed by mixing ampholytes of various pH ranges. (Concentrations of about 7---8% were found to be suitable for most applications.) Examples of equilibration solutions are as follows: Wide range gradient: 1.8 ml pH 3.5-10 ampholytes, 0.2 ml pH 4-6 ampholytes and 0.4 ml glycerol made up to 10 ml with distilled water. Narrow range gradient: 1.8 ml pH 4-6 ampholytes, 0.4 ml pH 5-7 ampholytes and 0.4 ml glycerol, made up to 10 ml with distilled water. The Cellogel strips were transferred directly from methanol to the ampholyte equilibration solution without allowing the medium to dry out. They were floated face down on the solution to soak up ampholytes for at least two hours before use, and preferably overnight. 4. Electrode solutions The anode solution was 0.2 M citric acid and the cathode solution was 0.2 M ethanolamine. In the U77 electrophoresis tank each compartment contained 400 ml of its corresponding solution. Methylated Cellogel wicks were used, impervious side down, on top of running Cellogel strips. 5. Recommended

method

The ampholyte-impregnated, methylated Cellogel membranes were transferred to the cooling platen, non-porous side down without blotting either side before transfer. Membranes were rolled gently from one edge in order to exclude any air bubbles and make good contact with the cooling platen. The surface solution was then removed by blotting very lightly with Whatman No. 1 filter paper. The wicks were overlaid at each end by 0.5-1.0 cm. The entire

185 TABLE I FALL

IN CURRENT

WITH

TIME

DURING

ISOELECTRIC

FOCUSSING

AT

A CONSTANT

500

VOLTS Time

mAmp

(mh)

0 15 30 45 60 75 90-120

5.7 X 14 cm, pH 3.5-10

5.7 X 14 cm, pH 4-6

4.5 3.5 3.0 2.6 2.0 1.8 1.5

2.5 1.8 1.5 1.3 1.2 1.1 1.1

running surface was well blotted with Whatman No. 1 filter paper. Membranes were ready to take samples at this stage. Sample treatment and applications. Serum samples were untreated, other protein samples were dissolved in l--2% ampholytes or dialysed against weak (0.45%) sodium chloride solutions. For permissible protein concentrations, see results. Samples were applied using a Cellogel microapplicator (0.8 ~1) across the mid-point of the strip keeping 1.0 cm clear of each edge. 5 Samples could be applied across the 5.7 cm strip, or 18 across the 17 cm strip. An isoelectric focussing marker could be used to follow the course of the separation (see Results). The anti-condensation lid was placed in position and cooling water was started. Conditions. Initial power settings are shown in Table I. Separations were run at 500 V with constant voltage and no further adjustment of power settings. Protein stain. Following completion of the run, membranes were washed in methanol/water (70 : 30, by vol.) to remove excess ampholytes before staining. They were then transferred to Coomassie Blue R, 0.15 g/d1 in methanol/water/ acetic acid (5 : 5 : 1, by vol.) for 10 min. Meth~ol/water/acetic acid (5 : 5 : 1, by vol.) was used for detaining. Strips could be made transparent if necessary by using the standard method for clearing Cellogel (30% diacetone alcohol in water). 6. fmmunofoeussing The example of the technique given here is for the detection of serum immunoglobulins, although it should be applicable to many other proteins. Sample dilution was required and this depended on the concentration of the myeloma protein and the antiserum employed. For example, myeloma proteins of 20-30 g/l required a serum dilution of l/20-1/40 for Boehring monospecific antisera, but only l/10-1/20 for Dako mono specific antisera. One undiluted and three diluted samples were focussed in the normal way on a modified Cellogel strip. On completion of the separation, the strip was cut into four portions len~hwise. The undiluted sample was taken for protein staining immediately, and the three diluted samples were immunofixed with specific antisera. This was accomplished by floating them face down in 0.1 ml

186

of the

corresponding anti I@, IgA or IgM an a glass plate for about fO min. &reacted proteins were then removed by shaking in physiolo~caI saline for at least 4 h before staining for proteins. A double antibody technique was used in some cases, by floating the strips on 0.05 ml goat anti-rabbit imm~l~oglob~~Ii~~s antiserum for 10 min and washing in physiological saline overnight before protein staining. Results and discussion

A. Experimental conditions (i) The medirtm. M~thylation

of free carboxyl

~uups

in the medium

was

#

1

3

Fig. 1. Isaelectric focussing of, from left to right, a protein marker mixture (see text and Fig. 6) and four runs of the same serum on a Cellogel membmne without any pretreatment. The membrane was pre~u~brat~ with a portion of the same ampbelyte solution PH 3.5-10 used for the separations shown in FM. 2 and 3. Fig. 2. Isoelectric focussing of, from left to mixture on a CeIlogel membrane transfened ampholytes pH 3.5-10.

right, four runs of the same serum, and a protein marker from 100% methanol. The PH gradient was formed using

Fig. 3. Isoelectric focussing of the samples shown in Fig. 2 an Cellogel membrane after methylation. The final PH gradient of the run was 3.&-9.0. See text for samples and amounts applied.

187

undertaken to minim&e electro-endosmosis which otherwise tended to move the established gradient slowly to the cathode. Without methylation, the gradient was not maintained stationary nor was the separation as well defined, as illustrated in Figs. l-3. These separations were run under identical conditions except that the Cellogel in Fig. 1 was non-methylated and stored in 30% methanol, in Fig. 2 it was non-methylated but stored in absolute methanol and in Fig. 3 the methylated product was used. The width of the methylated Cellogel membranes was found to be satisfactory up to the 17 cm size tried. The choice of width would obviously depend upon the application, narrow width 5.7 cm being easier to handle but the wider membrane being more suitable for multiple samples. The length of 10 cm was found to be optimum; longer strips did not produce as good separations. (ii) Ampholyte equilibrations. The minimum ampholyte concentration to effect a serum protein separation was found to be 8%. High ampholyte concentrations also tended to counteract electro-endosmosis in conjunction with methylation. Parts of the pH gradient, which were weak and which were prone to localized overheating, e.g. pH 4-6 were reinforced. A need to define the ends of the gradient depended upon the sample being analysed; the wide range Ampholines (pH 3.5-10) as supplied gave a gradient of about 3.6-9.0 with non-linear compression at the cathodal end, that is, at pH 8.0 and above. For serum proteins this was not important; a reproducible pattern was still obtained. However, for other proteins with high isoelectric points e.g. myoglobin, the effect can be removed by strengthening the end of the gradient with the appropriate narrow range ampholytes. Glycerol was included in the ampholyte equilibration mixture to give the Cellogel membranes better handling characteristics by helping to prevent them drying out. (iii) Electrode solutions. The electrode solutions described for the method were satisfactory for the range of pH gradients tried. They were inexpensive and caused little damage to the methylated Cellogel which was quickly deacetylated by stronger alkalis. The cathode wick could be used for several days without replacement, as could the electrode solutions. (iv) Voltage, power and cooling. Cooling was satisfactorily achieved using a water-cooled, plastic platen, although one constructed of glass might be more efficient. The environment within the tank was closed to the outside except for two small vent holes in one side of the tank. This seemed to be necessary to stop the strips drying out resulting in distortion of patterns. An anti-condensation lid was necessary because of the closed tank. Several observations were made to find optimum time and power requirements. Separations were effected at a constant 500 V which considerably simplified the procedure, although they could be started at a more cautious 300 V with a gradual increase in voltage throughout the run if required. The final current should be near 1 mAmp during the final stage of the separation. Poorly methylated strips had a final current of greater than 1 mAmp per 5 cm width, and also significant endosmosis. (v) Sample preparation and application. The best way of applying sample to the methylated Cellogel membranes was found to be by the double wire technique and particularly by the use of the Cellogel@ micro-applicator which

188

deposited 0.8-~1 samples. When using serum samples, a small amount of’ denatured protein might remain at the origin, If a particular protein is being studied, the point of a~~licatioI1 should be kept away from the final position of the protein as determiIled by trial. All samples should be applied along the same line if inter-sample comparison is required, It was found that the midpoint of the strip was usually satisfactory. Because of edge effects, 1.0 cm clearance from the edges was maintained. The total amount of protein should be adjusted sa that even minor components of interest could be detected. The sensitivity for individu~ proteins was influenced by affinity for dye binding. In some cases, e.g. albumin, the high ampholyte concentrations was found to interfere with staining if the prewash had not been carried out. With serum it was found that membranes prepared

6

4 Fig. 4, Isoelectric focussing of sera on methykted Cellogel membrane 4.O-S.5. See text for samples and amounts applied. Fig. 5. Isoelectric focussing of various non-serum left to right the samples are crude egg albumin, anhydrase.

using a narrow

range pH gradient

of

proteins (5 mg/ml) using a pH gradient 3.S9.0. From 90% egg albumin, catalase, haemoglobin and carbonic

Fig. 6. Isoelectric focusing p&I range 3.5-9.0 of several protein markers. Isoelectric points were assigned to the main protein zones obtained in each case. From Ieft to right, bovine serum albumin, LT-lactogiobulins (A + B), marker mixture, carbonic anhydrase and myogfobin (equine).

189

TABLE II ISOELECTRIC

POINTS OF THE PROTEIN MARKERS

Protein

Source

(ref. 4)

Isoelectric point (PI)

Bovine serum albumin @LactoglobuIin Carbonic anhydrase Myoglobin

Sigma A7511 Sigma L6879 Sigma c7500 Sigma MO630

4.93 5.35 5.45 6.18 7.58

for wide and narrow range gradients would take 2 X 0.8 ~1 sample. Concentrations of most other proteins of up to 20 mg/l could be tolerated if 0.891 samples were used. Relatively high concentrations of salt were tolerated because of the high ampholyte concentration. (vi) Staining. Although the proteins could be stained directly in the Coomassie Blue if required, the preferred technique was to remove the excess ampholytes first by a pre-wash. This shortened the destaining time considerably.

B. Protein separations (i) Serum proteins. The results in Fig. 3 show the wide pH gradient

separation of four O&p1 applications of the same normal serum. On the right of this Fig. 3 an isoelectric focussing marker mixture has also been run (to be described later). Although detail has been lost making the photograph good resolution of minute serum protein zones was observed. The zones of al-antitrypsin, the most anodic of serum proteins are clearly visible. Serum albumin because of its high concentration shows intense staining and some distortion. The results in Fig. 4 show several different serum samples separated using the narrow pH 4-6.5 gradient. Normal sera were run in position l-3 and positions 4 and 5 were occupied by the same serum from a child with cu,-antitrypsin deficiency phenotype Pi Z. Position 5 was the only double sample application (2 X 0.8 ~1). This gradient separation might be used to phenotype ar,-antitrypsin deficiencies in combination with control sera from known cases, as already carried out by isoelectric focussing on polyacrylamide gel [ 31. (ii) Other proteins. The results in Fig. 5 show the separation of several other proteins. From left to right is crude egg albumin, 90% pure egg albumin, catalase, carbonic anhydrase and haemoglobin all at 5 mg/ml and with 0.8 1.11 applications from the same start line. The method may be used to monitor stages of protein purification; compare, for example, crude egg albumin and 90% pure egg albumin. It may also be used to test the purity of individual proteins alone, or in conjunction with immunoprecipitation. (iii) Isoelectric focussing marker mixtures. The results in Fig. 6 show the separation of, from left to right, bovine serum albumin, /3-lactoglobulin (A + B), the combined marker mixture, carbonic anhydrase and myoglobin. All proteins were 5 mg/ml and 1 X 8 ~1 applications were made on the same start line. There are at least two advantages of running an isoelectric focussing marker

190

mixture with protein separations. One is that the separation may actually he seen to be occurring satisfactorily especially in conjunction with an added tracer dye to mark albumin. Another is that a very good idea may be had of the isoelectric points of proteins of interest. Pure marker proteins should be used, but in these experiments proteins obtained from Sigma were used and the isoelectric point was assigned to the major protein zone (see Table II). Accurate determination of isoelectric points of proteins would probably be better made using a sucrose density gradient stabilised column technique. However, a straight calibration line was obtained for isoelectric points against distances from the anode, as shown in Fig. 7, and so this method could be used to obtain approximate isoelectric points.

The small study reported here on the immunofocussing of myeloma proteins served to demonstrate several important factors: (i) The quality of the antiserum determined the dilution of the antigen. For example, using Behring monospecific antisera the serum had to be diluted l/20-1/40 for an IgG myeloma protein of 25 g/l. In order to stain the protein adequately, a double antibody technique than had to be used. For the Dako antiserum only a l/10 dilution of the protein was necessary and the use of a second antibody was not needed. To “freeze” a protein zone in this manner, it was imperative that a large antibody excess be present, despite the fact that focussed proteins represented a very high local concentration of antigen. Antibody-antigen complexes redissolved in antigen excess and diffusion of zones occurred with subsequent loss of resolution. (ii) Ampholytes even at concentrations as high as 8% did not appear to interfere with the immune fixation reaction making prewashing with buffer un-

1

I 2

1 3

I 4

I 5

1 6

I 7

I 8

I 9

I 10

Distance from anode Icmi

Fig. 7. The pH gradient from anode edge determined using the marker mixture in Fig, 6 to determine pH.

191

necessary. The technique was successful with antigens throughout a large pH range as evidenced by focussing a l/10 dilution of serum and using a Behring anti-whole human serum. Many immunofixed zones were apparent, after this treatment. (iii) Several myeloma proteins of different classes were examined by immunofocussing. It became apparent that not only could the class be identified but that apparently homogeneous proteins by electrophoresis were heterogeneous by this technique, and that this was confirmed by the use of mono specific antisera. Up to seven discrete protein zones with an IgA myeloma have been observed, and five with an IgG myeloma. (iv) Immunofocussing on Cellogel is a new dimension in protein separation; it is a direct technique which can supply specific information after isoelectric focussing. As such, it could have application in other fields such as typing bacterial antigens or in forensic work. D. Other possibilities Staining for isoenzymes following focussing would also be a powerful technique for identifying specific groups of proteins. It is quite possible that direct reactions could be devised for many enzymes provided ampholytes did not interfere in the reaction, or could be removed by washing after the enzyme had been fixed. Cellogel is a relatively neutral material for protein separations and no polymerising agents are left to react with proteins during separation, unlike polyacrylamide gels [ 51. Acknowledgements I thank Dr. Geoffrey Walker, Head of Department, for his help in preparing this manuscript and Mr. Dallas Simpson for the photography. References 1 2 3 4 5

Wrigley, C.W. (1968) Science Tools 15. 17 Vesterberg. 0. (1972) Biochim. Biophys. Acta 257.11 Hoffman, J.J.L. and Van Den Broek, W.G.M. (1977) Clin. Chim. Acta 75,233 Barb. J. (1973) Science Tools 20. 29 Radola. B.J. (1976) in Isoelectric Focussing (Catsimpoolas, N.. ed.), P. 120. Academic Press, New York

Isoelectric focussing of proteins on cellulose acetate gel membranes.

183 Clinica Chimica Acta, 85 (1978) 183-191 0 Elsevier/North-Holland Biomedical Press CGA 9304 ISOELECTRIC FOCUSSING GEL MEMBRANES OF PROTEINS ON...
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