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
BIOCHEMKAL
AND BlOPliYSlCAl
RESEARCli COMMUNICATIONS
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
BIOCHEMICAL
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).
453