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EXPERIMENTAL PATHOLOGY
AND PARASITOLOGY, 6/13, 2003
Bulgarian Academy of Sciences
An improved procedure for silver staining
of protein bands on polyacrylamide gels
S. ZACHARIEVA1, E. PANEVA2, J. YANEVA2
1
Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences, 1113
Sofia, Bulgaria
2
Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Abstract
A streamlined protocol that allows high sensitivity visualization of protein bands by silver staining is
reported. The entire process, from gel to record, can be accomplished within three hours. Comparing with
the classical Coomassie brilliant blue dying of proteins, the proposed silver staining procedure is faster,
clearer, more sensitive and easily performed. It allows detecting protein bands in polyacrylamide gels
within the range of 10 ng/mm2. The stain is applicable to various acrylamide gel thickness and gel
concentrations.
Key words: histone H1, polyacrylamide electrophoresis, proteins, silver stain.
Introduction
Silver stain technology was first applied
for histochemical purposes; ammoniacal
silver complexes have been used in histology
for a long time – to obtain fine definition
unobtainable by other methods. Later on
the approach was applied also for detection
of proteins and nucleic acids on polyacrylamide gels (S w i t z e r et al., 1979).
Especially for proteins, the original procedure was modified and improved repeatedly and it was firmly demonstrated to be
faster and more sensitive (M e r r i l et al.,
1981; M o r r i s s e y, 1981; S a m m o n s et
al., 1981; M o l d et al., 1983; N i e l s e n
and B r o w n, 1984; W i l l o u g h b y and
L a m b e r t, 1983; W r a y, 1983; K u r o s a k i et al., 1984; N i e l s e n and B r o w n ,
1984; B l u m et al., 1987; H i l b e r t et al.,
1995) than the classical Coomassie brilliant
blue staining (F a i r b a n k s et al., 1971).
Nowadays many investigators examine
protein samples only in extremely limited
amounts, i.e. during the process of isolation
and purification of individual fractions.
That is whay it is so important to utilize
staining with maximal sensitivity and
quickness. Recently we have described a
simplified protocol for DNA silver staining
in polyacrylamide gels (P a n e v a et al.,
2000). Here we proposed an adapted
version for imaging of protein bands on
polyacrylamide gels by silver staining: an
example with major nuclear protein histone
H1 and various protein markers. The procedure is fast, easy to handle and effective
for testing huge series of fractions during
the process of protein isolation and
purification.
35
Materials and Methods
Chemicals. All chemicals – acrylamide, N, N’-methylene-bisacrylamide
(bis), sodium dodecylsulphate
(SDS),
ammonium persulphate (APS), tetramethylenediamine (TEMED), Tris (hydroxymethylaminomethane), glycine, BPB
(bromphenol blue), Coomassie brilliant
blue R-250, silver nitrate and formaldehyde were of analytical grade and
purchased from Sigma (Saint Louis, MO,
USA).
Isolation and purification of histone
H1. The major nuclear protein – histone
H1 was purified from mouse liver nuclei
under non-denaturing conditions on CM
Sephadex C25 columns following the procedure described by B a n c h e v et al.,
1991. The concentration of histone H1
was determinated spectrophotometrically
using an extinction coefficient of 1.85 ml.
cm-1. mg-1 at 230 nm wave length.
Polyacrylamide electrophoresis under
denatured conditions. Electrophoresis was
performed in vertical polyacrylamide slab
gels (150 × 120 × 1 mm), containing 0.1%
SDS (L a e m m l i, 1970; S a m b r o o k
and R u s s e l, 2001). About 20 mm long
was the 6% stacking gel on the top of the
15 % separating gel (acrylamide to bisacrylamide ratio 29:1), containing 4.5%
Tris-HCl (pH 8.8), 0.1% SDS, 0.025%
ammonium persulphate, and 0.05%
TEMED. PAGE running buffer (pH 8.3),
contained 0.3% Tris, 1.44% glycine and
0.1% SDS. Usually the gels were first prerun for 30 min at 300V (constant) and 60
mA and then run with protein samples for
about two hours at 250 V and 50mA. All
the time plastic gloves were worn while
handling the gels.
Sample preparation. The protein samples (different amounts of histone H1 or
various marker proteins) were prepared in
sample buffer (pH 8.0) containing 5% glycerol, 0.121 % Tris, 1% SDS and 0.0292%
EDTA. The samples were heated in water
bath at 95oC for 3 min and allowed to cool
at room temperature. Then 0.1% bromp36
henol blue as a tracking dye was added to
each sample and they were layered into
gel slots using special loading tips. When
the tracking dye reached about 2 cm of
the bottom of the gel, the current was
turned off and the gel was removed.
Coomassie blue R-250 staining. Two
grams of Coomassie blue R-250 reagent
(Sigma, MO, USA; # B-0149, Lot
13H5002) were dissolved in 400 ml of
methanol; then 300 ml of deionized water
and 200 ml of 50% acetic acid were added
with continuing stirring to complete the
preparation (0.2% Coomassie brilliant
blue R-250 in 40% methanol – 10% acetic
acid). For Coomassie staining of one of
the two identical gels was placed in 150 ml
of this solution for four hours under slight
shaking. Then the gel was unstained with
10% methanol – 5% acetic acid with
many changes until the background became clear and the protein bands apparently
visible.
Silver staining of protein bands. The
whole protocol of the silver stain procedures with determinated standard conditions is presented in Table 1. The stock
solution of silver nitrate (Sigma, USA,
catalog number S-8157) has been prepared with deionized water and kept in dark
bottles at 4oC and used within 1-2 weeks.
Developer solution – formaldehyde
(cat.# F-1635) in sodium carbonate has
been prepared always just before use.
Protein silver-staining procedure. The
pictures of the stained gels were taken
with PRACTIKA MTL5 camera (objectglass 2.8/50) using back white light source.
When needed the gels were died between
two wet cellophane sheets at room temperature or in vacuum dryer and kept as
files for a long time.
Results
All single steps of the silver staining
procedure for detection of histone H1
bands after polyacrylamide electrophoresis are outlined in Table 1. The propo-
Table1. Main steps of the proposed protein silver-staining procedure
STEP
DURATION
AND TEMPERATURE
1. Fixing the gel in 50% methanol10% acetic acid.
2. Thorough washing with dH2O
3. Fixation the gel in 1% nitric acid
4. Rinsing twice with dH2O.
5. Soaking for impregnation
in 0.3%AgNO3.
6. Rinsing briefly with dH2O
7. Developing in 2.5% NaCO3 +
75ìl 37% formaldehyde in
100ml dH2O prepared just
before use.
8. Rinsing twice with dH2O.
9. Taking a picture using back
illumination of white light.
10. Fixation in 20% methanol5 % glycerol
11. Rinsing with H2O.
12. Drying the gel between two
wet cellophan sheets at
13. Keeping as a record.
sed procedure represents a compilation of
our previous and other authors’ protocols
(S w i t z e r et al., 1979; M e r r i l et al.,
1981; M o r r i s s e y, 1981; S a m m o n s
et al., 1981; M o l d et al., 1983; N i e l s e n and B r o w n, 1984; W r a y, 1983;
K u r o s a k i et al., 1984; N i e l s e n and
B r o w n, 1984; B l u m et al., 1987;
H i l b e r t et al., 1995; P a n e v a et al.,
2000). Since 1990 we have been investigated the interaction of the linker histones
(H1, H5, H1° and cognates) with DNA
(Y a n e v a et al., 1990; Y a n e v a and
Z l a t a n o v a, 1991; Y a n e v a et al.,
1995; Z l a t a n o v a et al., 1995; Y a n e v a et al., 1997). Many observations point
to the involvement of linker histones (H1,
its subtypes and variants) in the switching
mechanisms that determine gene expression (B u t t i n e l l i et al., 1999). The
importance of these investigations often
forced us to isolate, purify and analyze
simultaneously variuos types of linker
histones from different mammalian tissues. For direct comparison two identical
NOTES
2 hours, r.t.
up to 4 hours
3 times, 2 min each
20 min
1-2 min each
20 min, r.t.
slight shaking
1-2 min, r.t.
10-12 min
up to appearing of
brownish bands
1-2 min each
15-20 sec
1-2 h, r.t.
2-3 min
o.n., 23-26oC
up to 48 hours
forever
polyacrylamide gels with decreasing amounts of histone H1 were stained either with
silver stain and Coomassie brilliant blue
stain (fig. 1). It is well visible that the
protein bands on silver stained gel (fig. 1,
A) are quite more prominent than the
same on the identical Coomassie brilliant
blue stained gel (fig. 1, B). On the first gel
the bands are apparently visible up to lane
8 (50ng of the protein sample), but on the
second gel – up to the forth (3ìg sample).
The same protocol for silver staining was
applied on another protein samples –
various protein markers (Sigma, USA) –
with different molecular weights (fig. 2).
We have avoided an overnight fixing in
methanol-acetic acid because separated
experiments with isotope labelled proteins
showed decreasing of the silver stain
sensitivity by 20% (unpublished observations). Sometimes several portions of
methanol-acetic acid were useful for initial fixing of the separated proteins on
PAAG. The consequence was a satisfactory reduction in background.
37
À
Â
1
1
2
2
3
4
3
4
5
5
6
7
6
7
8
Fig. 1. Silver staining of the major nuclear protein histone H1 on PAAG (A) compared with
Coomassie - stained one (B). Decreasing amounts in micrograms of purified mouse liver
histone H1 were loaded onto similar lanes of two identical 15% polyacrylamide gels
containing 0.1% SDS: 10ìg (lane 1); 5ìg (2); 4ìg (3); 3ìg (4); 2ìg (5); 1ìg (6) ; 0.5ìg
(7); 50ng (8). Note that the protein bands on the gel obtained after silver stain are visible
up to lane 8 (50ng) while after Coomassie blue stain – only up to lane 4 (3 ìg)
38
phosphorylase (rabbit muscle) - 97.4 KD
bovine serum albumin - 66.0 KD
albumin (chicken egg) - 45.9 KD
carbonic anhydrase - 29.0 KD
β - lactoglobulin - 18.4 KD
top
bottom
a - lactoglobulin - 14.2
KD
histone H1AB histone H1ο
Fig. 2. Another example with silver staining – mixture of various marker
proteins with different molecular weights -500 ng of each sample (denoted
above) and histone H1 – subfractions obtained after 12% polyacrylamide
electrophoresis (according to Laemmli, 1970)
Discussion
The exact mechanism of the silver stain
reaction is still unclear. Probably it involves the formation of initial nucleation
sites at certain charged amino acid side
chains of the proteins, followed by the
rapid buildup of silver deposits in the
presence of a reducing agent (usually
formaldehyde) from free silver ions in the
gel or in solution. The deposition of silver
ions is allowed to continue until protein
bands become visible and the background
is unacceptable (M o l d et al., 1983;
K u r o s a k i et al., 1984; H i l b e r t et
al., 1995). The main drawbacks of most
protocols for protein silver staining have
been the long duration and an unspecific
background staining. Here we present a
quick and quite sensitive procedure for
imaging of protein bands by silver stain
avoiding that drawbacks and obtaining a
transparent background. The process
might be completed effectively within
three hours. This prescription allows to
detect proteins in PAAG within the range
of 10 ng/mm2. It should be mentioned that
the proposed procedure is especially
suitable for the cases of fast testing of the
column fractions during protein isolation
and purification.
Abbreviations used: AA, acrylamide;
APS, ammonium persulphate; KD, kiloDaltons; o.n., over night; PAAG, polyacrylamide gel; r. t., room temperature; SDS,
sodium dodecyl sulphate; TEMED, tetramethylenediamine; Tris –hydroxymethylaminomethane
39
Acknowledgements
This work was supported by a research
grant (K1003/00 to J.Y.) from the Bulgarian Ministry of Education and Science.
The excellent photo service of Mr. Z.
Apostolov during the experiments is also
appreciated.
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Received 10.12.2003