Polyacrylamide-isoelectric-focusing a new technique for the electrophoresis of proteins

Vol. 32, No. 3, 1968

POLYACRYLAMIDE-ISOELECTRICET)CUSING A NEWTECHNIQUE FORTREELECTROPHORESLS OF PROTEIbls D.H. Leaback and A.C. Rutter Biochemistry Department, Institute of Orthopaedics, Stanmore, Middlesex, England.

ReceivedJune 24, 1968 In this new technique, proteins migrate through a pH gradient to equilibrium

positions within a slab of large-pore polyacrylamide

The pR gradient is established, (*AmpholinesS, L.K.B. electric

field.

application existing

Barr,

Xnstrueents Ltd.)

The technique offers

and high resolving-power

Tuttle,

and, in addition,

19%;

the

advantages

of the applied of sample

(ease

dependent only upon protein

charge)

principle

of

(Hooh and

Svensson, 1962; Vesterberg and Svensson, 1966)

employs simple apparatus, co5serves expensive carrier-

ampholytes, is very resistant

to

convective

mixing, and permits the

simultaneous separation of several mixtures. protein,

ampholytes

under the influence

the *isoelectric-focusing'

procedures utilising

1955;

and maintained, by carrier

gel.

subjected to densitometric

The gelsup

analysis and

be stained for

for storage.

dried

Exmaplee have been described of the use of the tachnique for the resolution of comp.lex protein mixtures, of the isoelectric

points of proteins and for

Since a preliaimary Rutter,

the separation of isoenaymes, the deterrinstios

account

rob

purposes.

of this merk mae presented (Leaback md

19681, a note has appeared concerning

in polyacrylamide

preparative

@ale and Iatner,

a similar

procedure conducted

19681.

Methods The apparatus devised for this mark (Fig. bearing terminals (T) conmected to horizontal

447

1) comprises a perspex lid

carbon

or platinum electrodes

(El,

Vol. 32, No. 3, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BASE

Fig. 1.

and a glass base to carry the gel (G) and the electrode wells (A, B). only other major requirement is for a power eupply of &~O

Volta

The base consists of a glass plate to which four strips

o-3mA.

of

glass

are

epoxy-resin)

A perapex strip

cm? is placed at A and another at B before the well

is filled

with polymeriaation

0.858. acrylamide, ethylene-diamine 1.43g. acrylamide,

mixture (either

a rigid

and 2Omg. ammoniumpersulphate or, for 75mg. NN'-methylene-bis-acrylamide,

After

setting,

cm!

25pl. NNNk'-tetramethylphotopolymeriaation, 0.04mg. riboflavin

40%Ampholine solution)

and is overlaid

perspex sheet bearing moulds for sample-slots (five

are convenient).

18~8~0~2

JOml. of solution containing

5Omg. NN'-methylene-bis-acrylamide,

and O.J-0.61~1. of the appropriate

well

D.C. at

bonded (with 'Araldite' 8x2x0.2

to form a water-tight

The

peraulphate-polymerized

with 2-5 changes of water before the appropriate (0.5-1.01111.) is spread upon the gel surface and

by

slots lOxlxlmm! gels are washed

24%Ampholine solution allowed

to

stand at room

temperature for several hours before use. Electrode acid (0.M)

wells

A and B are filled

with aqueous solutions of phosphoric

and diamino-ethane (0.15H) respectively,

448

the samples are added to

Vol. 32, No. 3, 1968

BIOCHEMICAL

slots S and, with the lid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a potential

in position,

difference

the electrodes such as to keep the current at or below 2mA. prevents excessive evaporation

is applied to A moist pad (C)

At field

from the gel surface.

strengths of

20-25V./cm. homogeneousproteins give sharp bands at room temperature whereas ampholytes of low molecular weight (e.g. methyl red, haematoporphyrin) require field

strengths over 7OV./cm. for bands of comparable sharpness.

Whenequilibrium

the gel (and glass base) is immersed in

is attained,

aqueous trichloroacetic Since carrier

acid (5% w/v). ampholytes complex with protein dyes, the gels were washed

several times in the trichloroacetic

solution before staining

0.05% Naphthalene Black in methanol;glycerol;water;acetic vol.),

and subsequent destaining in aqueous acetic

densitometric

acid;50;50;20;1

(7%/v).

by

For storage or

analysis the fixed gels were washed several times with 7% (v/v)

aqueous acetic acid, spread upon cellulose Washed'persulphate'

tri-acetate

sheets and air-dried.

gels were used throughout the results presented

here to obviate the possibility

of protein interactions

the polymerisation

Mitchell,

mixture (cf.

experienced in obtaining reproducible isation

(3Omin. with

with components of

and since difficulties

1967)

large-pore

were

gels (3%) by the photopolymer-

procedure. Endosmosisin gels prepared as above was low (1-2cm./16hr.).

contrast,

experiments with strips

and Flodin,

of paper, methylated paper (cf.

1954) or cellulose acetate indicated

materials precludes the

In Tiselius

that the endosmosis on these

of adequately stable pH gradients.

establishment

Results The technique has been applied to the separation of the common haemoglobins A, C, F and S. oxy- forms

(Figs.

bands after

3-6

These proteins,

2a, 2c and 2d respectively) hr. electrophoresis

as the met-, carbonmonoxy-, or separate as sharp, well-resolved

in a 6-8~~ gradient.

the separation of oxyhaemoglobins A and F in particular

449

The discreteness of indicates

that the

Vol. 32,No. 3, 1960

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AND BlOPliYSlCAl

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2. The portions of gels shown here correspond approximately to pR 6.5-8.0 (left to right) with mixtures of met- (a and. b), carbonmonoxy- (c),

Fig.

and oxy- (d and e), forms of the haemoglobins indicated ? were of unknown species.

technique has advantages over cellulose acetate (Briere, Ratsakis, 1965) or polyacrylamide

electrophoresis

above.

Bands marked

Golias and

(Dowding and Tarnoky, 19671.

By using carbonmonoxy-haemoglobins A and C as marker proteins of known characteristics

(cf.

Svensson, 19621, the iso-electric

points of carbonmonoxy-

haemoglobins F and S have been estimated from gel measurementsto be 7.07 and 7.23 respectively. The use of the technique to follow the chemical modification protein

was investigated

by labelling

method of Rinderknecht (1962);

of a

bovine serum albumin according to the

the introduction

450

of the fluorescein

moiety

8lOCHEMlCAL

Vol. 32, No. 3, 1968

brought

about

point F (Fig.

AND BIOPHYSKAL

a marked acid-shift 3(b)).

in the principal

The location

of erythrocyte

spraying gels with 4-methylumbelliferyl (Fig. 3(d)) gave multiple

protein band to the acid phosphatases after

phosphate and exposure to ammonia

fluorescent

bands (A) which appeared to be

sharper than those described for a starch-gel & Harris,

RESEARCH COMMUNKATIONS

procedure (Hopkinson, Spencer

1964).

Zones have been assigned for someimportant components of a typical humanserum (Fig. J(k)) Pharmaceuticals Ltd.) transferrins, of specific

using authentic

purified

proteins

as marker substances (e.g. haptoglobins,

Fig. 3j>; localisation

more detailed assignments will

Fig. 3i,

follow the application

techniques.

Since protein bands sharpen as equilibrium

is reached, very minor

components of protein mixtures can often be detected. of haemoglobin A2 can readily haemoglobin A (Fig.

(Hoechst

Thus, a few percent

be demonstrated in the presence

Z(e)) and minor components in crystalline

excess

of

samples of

ovalbumin (Sigma Chemical Co.), bovine serum albumin (Armour Pharmaceutical Co.) and bovine pancreatic (Figs. 3(a),

ribonuclease (British

3(b) and 3(c)

Drug Houses) have been detected

respectively).

Advantage may be taken of the procedure for re-running protein

ease

bands.

of sample application

in a

Thus, lcm. squares were excised

from a gel containing a series of bands (Fig.

3(e)) obtained from a human

immunoglobulin G (homogeneousto immune-electrophoresis);

the squares were

placed in lcm? holes in another gel and the proteins re-run to give the patterns shown in Figs. 3(f),

3(g) and 3(h).

It was concluded that the banding

in Fig. 3(e) represented true molecular heterogeneity complexing of an homogeneousprotein

with various Ampholine species.

In the column procedure of Svensson (1962) gradient employed is only partly sample overloading, fractions.

and not, for example, the

the sucrose concentration

successful in opposing mixing due to

to convection and to turbulence while drawing off the

The technique in polyacrylamide

451

gel is very resistant

to

Vol. 32, No. 3, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 3. The portions of gels shown here correspond approximately to pH3-10 (left to right) with (a> ovalbumin, (b) bovine Serum albumin, (c) ribonuclease, cd> drawing of erythrocyte phosphatase activities (A) with oxyhaemoglobin A indicated at position B, (e) human )'-globulins, (f), (g) and (h) fractions of (e>, (i> humanhaptoglobins, (j) humantransferrins and (k) a 'normal' humanserum with prealbumin (A), haptoglobin (B), albumin (Cl, transferrin (D) Sample slots are marked S. and Y-globulin (E) zonea.

452

Vol. 32, No. 3, 1968

convective

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mixing and to sample overloading.

Furthermore, the high resolving-

power of the method can be preserved by excising appropriate gels (colourless

protein bands being located with respect to coloured

marker ampholytes).

Another technique has been used successfully

the necessity of extracting

gel segments for preparative

the sample is applied to a 7Ox2x3mm!slot (S) and, after carried by endosmosis into another such slot (slightly of the initial

portions of the

equilibrium

position)

necessary, replaced by carrier

purposes.

to avoid Thus,

sharpening, bands are towards the cathode end

where the protein is removed and, if

ampholyte and the process repeated.

As much

as 3Omg. of a haemoglobin A/A2 mixture have been applied to a single gel and separated as electrophoretically-pure The technique offers

potential

components by this procedure. for a wide range of routine and

research applications. References Briere,

R.O., Golias, T., and Batsakis, J.G., 695,

Amer. J. Clin. Path.,

44,

(1965).

Dale, G., and Latner, A.L., The Lancet, 1, 847, (1968). Dowding, B., and Tarnoky, A.L., Proc. Ass. Clin. Biochem., k, 167, (1967). Hoch, H., and Barr, G.H., Science, 122, 243, (1955). Hopkinson, D.A., Spencer, N., and Harris, H., HumanGenetics, l.6, 141, (19641. Leaback, D.H., and Rutter, A.C., Proc. Biochem. Sot., 481st Meeting, April 18, 1968. Mitchell, WM., Biochem. Biophys. Acta., 141, 171, (1967). Rinderknecht, H., Nature, z, 167, (1962r Svensson, H., Arch. Biochem. Biophys. Suppl., 1, p.132, (1962). Tiselius, A., and Flodin, P., Adv. Protein Chem., 8, 461, (1954). Tuttle, A.H., J. Lab. Clin. Med., 5, 811, (1956). Vesterberg, O., and Svensson, H., Acta. Chem. Stand., 20, 820, (1966).

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