Download Simplified silver protein detection and image

Electrophoresis 1982,3, 17-23
Simplified silver stain
Equation (44) becomes
r l $f
<
r2 4;
(46)
or
(47)
Equation (47) is of the form used by Jovin (equation 65 of
[91) to state general steady-state conditions for moving boundaries. This expression accounts for the change of constituent mobilities of weak electrolytes, when the pHs of
phases on either side of the boundary are unequal. Since
4 = 1 for strong electrolytes by definition, equation (47)
reduces to:
rl <r2
Carl R. Merril,
David Goldmano and
Margaret L. Van KeurenO
Laboratory of General and
Comparative Biochemistry,
Laboratory of Clinical Sciencen and
National Institute of Alcohol and
Alcoholismo, NIH, Bethesda, MD
(48)
17
In equations (38) to (48), the mobility of anions is being considered, and mobilities carry the same sign as the charge on
the ion. The species with the more negative value of mobility
therefore has the higher mobility.
The question of whether the boundaries are at the steady
state is left out of most ITP treatments. The steady-state conditions for weak electrolyte boundaries are discussed by
Alberty [161 and Jovin [91, and are given in Appendix 11.
As to the SNEP and TEP systems of Schafer-Nielsen and
Svendsen, a reading of the papers of Svensson [ 151, Alberty
[161, Dismukes and Alberty [171 and Nichol [211 should
clarify the fact that these authors have simply restated the
more general case of moving boundaries of weak
electrolvtes. Calculations of phase compositions and boundary velocities are explicitly considered in these references.
Simplified silver protein detection and image
enhancement methods in polyacrylamide gels
The use of silver for detection of protein in polyacrylamide gels is becoming
widespread. An effort has been made to develop a silver stain which minimizes the
time to perform the stain, the amount of silver used, and the complexity of the
procedures, and which maximizes the sensitivity. This stain is almost 200-fold more
sensitive than the Coomassie Brilliant Blue R-250 stain. This silver stain procedure
has been shown, with eight purified proteins, to generally be linear over a 40-fold
range in concentration, between 0.005 ng/mmz to 2.0 ng/mm2. For greater sensitivity, a recycling procedure has been developed. This procedure is capable of
amplifying trace proteins which could not be visualized with previous silver stain
techniques. Analysis of the kinetics of image development revealed that the silver
ion was the limiting factor in image formation. By recycling the gel through silver
nitrate solutions, silver ions are replenished in the gel, permitting amplification of
further details when the gel is treated with a developing solution. A number of
problems inherent in silver stain detection of proteins in polyacrylamide gels are discussed with suggested remedies.
take hours to perform, use considerable quantities of silver or
other expensive reagents, and give variable results. Since
The use of silver to develop images was discovered in the both histological silver stains and photochemical methods
mid-seventeenth century. The first application, the develop- rely on the selective reduction of silver, experiments were
ment of photography, was followed closely by the use of conducted to test the application of photochemical technisilver as a stain to visualize specific structures in histological ques to the problem of protein detection in polyacrylamide
specimens. Both fields now have long histories of empirical gels (PAGE gels). By soaking PAGE gels in silver nitrate, in
and often parallel development. Recently, the use of silver the dark, followed by use of common photographic dehas been adapted to detect proteins after polyacrylamide gel velopers, it was found that a negative image of the protein
electrophoresis (PAGE), first by adapting histological pattern may be obtained [71. Proteins are present in clear
methods [l-61 and then by employing photochemical regions of the electrophoretogram. Exposure of this image to
procedures 17-91. In general the histologically derived stains light results in some photoreversal. This method was not very
sensitive. However, by utilizing chemical photoreversal
procedures and formaldehyde as the image-developing
Correspondence: Dr. Carl R. Merril, Laboratory of General and Compaagent, a highly sensitive silver stain was developed [7-91.
1 Introduction
rative Biochemistry, National Institute of Mental Health, Bethesda, Maryland 20205, USA
Abbreviations: PAGE: Polyacrylamide gel electrophoresis; SDS: Sodium
dodecyl sulfate; TCA: Trichloroacetic acid
0 Verlag Chemie GmbH, D-6940 Weinheim, 1982
These photochemical silver stains have proven to be more
than 100-fold more sensitive than Coomassie Brilliant Blue
R-250 stains [8, 91. The procedures described in this study
0 173-0835/82/0 102-00 17S2.50/0
18
C. R. Merril. D. Goldman and M. L. Van Keuren
represent efforts to minimize the use of expensive reagents
and time, and to simplify the process while maintaining maximal sensitivity. During the development of these procedures, it was discovered that sensitivity can be limited by the
depletion of silver ions in the gel during image development.
By recycling the gel through silver nitrate, silver ions can be
replenished, and additional image details can be revealed
during subsequent development steps. This recycling procedure can achieve extremely high sensitivity.
Electrophoresis 1982.3, 17-23
model IP5000) and a PDP 11/60 computer (Digital Equipment).
2.5 Standard proteins and cellular proteins
Protein standards (Pharmacia) were phosphorylase B,
bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, ferritin, catalase, and lactate dehydrogenase. f14C] Proteins were cytochrome C (NEC 716,
23.6 ci/g), albumin (NEC 719, 20.0 ci/g), carbonic
anhydrase (NEC 718, 10.8 ci/g), ovalbumin (NEC 717,9.94
Ci/g) and phosphorylase B (NEC 727,9.0 Ci/g). E. coli pro2 Materials and Methods
teins were obtained from bacteria grown in M56 minimal
media supplemented with amino acids and glucose. E. coli
2.1 Chemicals
were grown to a density of 2 x lo8 celldm1 and then
centrifuged 2 rnin at 12500 x g. The supernatant was disAcrylamide, N,N'-methylenebisacrylamide, sodium dode- carded and the pellet resuspended in an equal volume of procyl sulfate (SDS), N,N,N',N'-tetramethylethylenediaminetein solubilization solution (see section 2.2) and heated to
(TEMED), ammonium persulfate, Biolyte carrier ampholy- 95 O for 5 min. The mixture was chilled on ice and again
tes and agarose were purchased from Bio-Rad. Coomassie centrifuged at 12500 x g for 2 min. The supernatant was
Brilliant Blue R-250 was obtained from Bio-Kad and Serva. stored at -70 "C. Mouse cells were grown in 75 ml flasks in
2-Mercaptoethanol was from Eastman Kodak as were the Eagle's Minimal Essential Media and harvested by rinsing
X-ray films and film developing chemicals. Tris base and twice with saline, followed by scraping with a rubber
glycine were from Sigma. Silver nitrate and anhydrous policeman. The suspended cells were centrifuged 10 rnin at
sodium carbonate were obtained from J. T. Baker. Purified 800 x g and the supernatant discarded. The mouse cell pellet
methylated [l4C1 proteins were purchased from New Eng- was treated as described for the E. coli cell pellet. Cell lysate
land Nuclear. Purified low and high molecular weight mark- protein concentrations were determined by the method of
er protein kits were obtained from Pharmacia. DNA restric- Lowry et al. [ 111.
tion fragments were purchased from Bethesda Research
Laboratories.
2.6 Photochemical silver stain
2.2 Two-dimensional electrophoresis
PAGE gels may be fixed for 20 rnin in either 2 0 % w/v
trichloroacetic
acid (TCA) or a solution of 50% v/v
Two-dimensional electrophoresis was performed according
% v/v acetic acid. TCA is recommended
methanol
and
12
to O'Farrell 101, using isoelectric focusing (4: 1 mixture of
0,5 mm. Gels are then washed three times
for
gels
thinner
than
carrier ampholytes of pH range 5-7 and 3-10) in the first
for
10
rnin
each
with
a solution containing 10 % ethanol and
dimension and electrophoresis through a uniform gel of 10 %
5
%
acetic
acid
(v/v).
The following steps are best performed
acrylamide in the second dimension. Protein samples were
or
amber
light to minimize background
under
subdued
solubilized in a solution containing 2 % w/v SDS, 5 % v/v
mercaptoethanol, 2 0 % v/v glycerol, 2 % v/v carrier am- darkening. Gels are soaked for 5 rnin in a solution containing
0.0034 M potassium dichromate and 0.0032 N nitric acid,
pholytes, pH 3-10, and 2 % v/v Triton X-100.
followed by 0.012 M silver nitrate for 20 min. The gels are
then rinsed with agitation in a solution containing 0.28 M
2.3 Autoradiography
sodium carbonate and 0.5 ml of formaldehyde (37 % commercial formaldehyde) per liter. This last step requires at
Gels were soaked 5 min in 3 % glycerol to prevent cracking least two changes of the solution to prevent precipitated
and then placed in a gel dryer (Model 224 gel slab dryer, Bio- silver salts from adsorbing to the surface of the gel. DevelopRad) under vacuum and mild heat. The dried gels were ex- ment is usually stopped, when a slightly yellowish backposed to Kodak AR-2 X-ray film in X-ray film holders con- ground appears, with a solution of 1 % v/v acetic acid for 5
taining intensifying screens. Exposure was at -70 OC.
min. Gels are washed twice for 10 rnin with water. They may
be stored in water or soaked in 3 % glycerol for 10 rnin and
then
dried between dialysis membrane (Bio-Rad) under a
2.4 Quantitative analysis
vacuum at 80-82 OC for 3 h. This latter procedure results in
Quantitation was performed by photographing stained gels a transparency which is relatively permanent. Some staining
next to calibrated photographic density standards (from the parameters must be adjusted for gel thickness. Reagent conNational Bureau of Standards) with 120 mm Tri-X film centrations and procedure times given above have been op(Eastman Kodak). These photographic images were scanned timized for 0.8 mm thick PAGE gels. Thicker gels require
at a resolution of lOOym with an Optronics 1000 HS scan- more time for each step.
ning densitometer at the 0-2 optical density setting. Image
densities were converted to optical density units using the 2.7 Recycling the silver stain
calibrated density standards to normalize images for variations in photography and densitometry. Analysis was per- The density of the silver deposited in each band or spot
formed with an image processor (DeAnza Systems Inc., reaches a maximum in about 15 rnin in the sodium car-
Simplified silver stain
Electrophoresis 1982. 3. 17-23
bonate/formaldehyde solution with gels 0.8 mm thick. Addition of fresh sodium carbonate/formaldehyde will not
enhance the density of the stained proteins at this point.
However, if the silver nitrate is replenished, additional image
density can be achieved. To achieve this image intensification, gels are stained as described above (see section 2.6);
however, development during the first cycle is stopped just
prior to development of a yellowish background with 3 %
acetic acid (v/v). Gels are rinsed twice with additional 3 %
acetic acid and then soaked for 10 min in fresh 0.012 Msilver
nitrate. The image is intensified by rinsing the gel with
sodium carbonate/formaldehyde as described (section 2.6).
If proteins require further staining, further recycling can be
performed.
2.8 Destaining
19
Brilliant Blue R-250 and/or silver. Gels stained with
Coomassie Brilliant Blue R-250 may subsequently be stained
with silver with no loss in silver-staining sensitivity. Gels
were photographed after each stain and densitometric
analysis was perfomed. The Coomassie Blue stains tested included W (Serva), G (Serva), R (Serva and Bio-Rad), and
Violet 49 (Serva). Greatest sensitivity was obtained by using
Coomassie Blue R overnight; this allowed detection of a
phosphorylase B band containing 0.5 pg of the protein or 3.7
ng/mm2. Photochemical silver staining allowed the detection
of a band of phosphorylase B containing 0.0027 pg or 0.02
ng/mm2. The silver stain is almost 200-fold more sensitive
than Coomassie Brilliant Blue R-250 for phosphorylase B,
albumin and ovalbumin.
3.2 Quantitative use
Destaining is accomplished by mixing equal parts of stock
solutions “A” and “B” and soaking the gel in the resulting
mixture until the desired destaining is achieved [31. “A” is
made by dissolving 37 g of sodium chloride and 37 g of
cupric sulfate in 850 ml of deionized water. Concentrated
ammonium hydroxide is added until the precipitate first
formed is completely dissolved and the deep blue solution is
adjusted to 1 liter. “B” contains 436 g of sodium thiosulfate
per liter. As it is difficult to control the rate of destaining,
photographs of stained electrophoretograms are made prior
to and during destaining. The rate of destaining may be
slowed by diluting the destaining solution. A 1:10 dilution is
suggested.
3 Results and discussion
3.1 Sensitivity
Fig. 1 compares three proteins stained with Coomassie
Brilliant Blue R-250 and the photochemical silver stain. The
purified proteins were diluted prior to electrophoresis so that
they were equal in concentration. The use of [l4C1proteins of
known specific radioactivity facilitated these adjustments.
Following electrophoresis, gels were stained with Coomassie
Phosphorylase
i
il
Fig. 2 and Table 1 show that the linearity of the silver stain
generally extends over a 40-fold range of concentration for
eight purified proteins. These purified proteins were electrophoresed in 10 % acrylamide slab gels which were sequentially photographed during development of the image in the
sodium carbonate/formaldehyde solution. The data shown
were obtained after 9 min of image development. We have
found that the stain becomes nonlinear when proteins are in
concentration greater than 2 ng/mm2. However, the range of
linearity can be extended by photographing at earlier times
during image development. In this manner, protein images
may be recorded before the staining of concentrated bands or
spots becomes nonproportional or saturating. Poehling et al.
[ 121 state that, “silver does not stoichiometrically stain proteins, unlike Coomassie Blue.” They measured peak areas
for albumin, lactalbumin, and carbonic anhydrase and concluded that while Coomassie Brilliant Blue R-250 staining
was stoichiometric from 10-200 ng, a 20-fold range of concentration, silver staining had “only a limited linearity
between 1 and 30 ng,” a 30-fold range. Examination of their
silver stain data, for which the linear portion of the protein
versus peak area curve was split and plotted in two separate
figures, reveals that silver staining for albumin and lactalbumin was linear from 0.5 to 15 ng, and carbonic
anhydrase was linear over a larger than 30-fold range. This
B
Albumin
n
Ovalbumin
1
Figure 1 . Densitometric comparison of Coomassie Blue and silver staining ofthree purified
proteins. The [14C1proteins were denatured
with SDS and mercaptoethanol, diluted in
20 % glycerol and electrophoresed in 10 %
acrylamide at 20 mA/gel. Gels were stained
E
A
i
with silver as described (see section 2.6) or
20
30
40
50
O 10
overnight with Coomassie Brilliant Blue R-250
DISTANCE ( c m l
(Bio-Rad). Gels were photographed and then
dried. Protein concentrations were verified with autoradiography, which was also used to measure protein band areas. Photographic negatives and
autoradiograms were scauned with a microdensitometer and density plots were made with the aid of a computer. The only band that could be visualized,
or measured, with Coomassie Blue staining was the 0.5 yg band (3.7 ng/mm2) of phosphorylase B (tracing E). Tracings A-D represent measurements
made from gels stained with silver. Each of the three proteins was present in the following concentrations: A = 0.5 yg, B = 0.055 pg, C = 0.027 pg and D
= 0.0027 ug. The limit of silver stain sensitivity for each of the proteins, calculated as ng of protein per mm‘, was 0.02,O. 18 and 0.40 for phosphorylase
B, albumin and ovalbumin, respectively.
L+
20
C. R. Merril, D. Goldman and M. L. Van Keuren
Electrophoresis 1982, 3, 17-23
data is fairly consistent with our experience with silver stains
(see Fig. 2 and Table 1). Quantitative use of the silver stain
requires care. Saturation of staining in protein bands or spots
results in underestimation of quantity. If the stains are
modified to enhance the production of “colored spots,”
radically different staining slopes are produced for different
proteins.
DENS1
?(
The slope of silver staining (densityhg protein) is probably
characteristic for each protein (Fig. 2). The slope for
ovalbumin is almost nine times that for carbonic anhydrase.
Even with different methods of silver staining, similar slope
ratios are obtained. The data of Poehling et al. [121 give a
ratio of albumin to carbonic anhydrase slopes of 4.1 when
analyzed for the linear part of the curve. Our ratio of albumin
to carbonic anhydrase slopes was 3.9 when density was plotted against ng/mm2 or 3.0 when plotted against ng. When
slope ratios of proteins are followed during image development, they are seen to be fairly constant (see Table 2).
11
1
1
ANHYDRASE
PROTEIN
nglmm‘
Figure 2. Silver image density versus concentration for eight purified proteins. Purified proteins (Pharmacia) were diluted in 20 % glycerol and
electrophoresed at 20 mA/gel. The gel (10 % polyacrylamide and 0.8 mm
thick) was stained with silver (see section 2.6), except that the gel was illuminated during development in sodium carbonate/ formaldehyde so that
it could be photographed at timed intervals. The data shown were obtained after 9 min in sodium carbonate/formaldehyde. Band area and density
for each protein were measured by computerized microdensitometry. Protein concentrations are expressed as ng/mm2, while density units are optical density times mm2.
Quantitative intergel comparisons using the silver stain are
possible if constitutive or marker proteins are present so that
other protein densities can later be normalized. Equivalent
amounts of protein should be loaded onto each gel. However,
since image intensity can be controlled by varying the length
of image development in sodium carbonate/formaldehyde,
differences in density may be minimized. To show that gels
may be compared despite a 10-fold variation in initial protein
loading, 10-200 pg of lymphocyte lysate was separated by
two-dimensional electrophoresis and stained. Densities of 45
protein spots on each gel were measured, and linear regression analysis was performed to compare the effects of varying protein loading and staining. Correlation coefficients
were 2 0.90 for all comparisons of protein loads from
10-100 pg (Table 3).
3.3 Image intensification and destaining
Fig. 3 shows a two-dimensional electrophoretogram of an E.
coli lysate which has been recycled through silver nitrate and
sodium carbonate/formaldehyde to intensify the image (see
section 2.7). Recycling permits the visualization of trace proteins which otherwise may not be detected. However,
background darkening becomes a problem with continued
recycling (see Table 4). Image intensification by recycling
Table 1. Linearity of silver staining for eight purified proteins
Proteina)
Albumin
Ovalbumin
Trypsin inhibitor
Ferritin
Carbonic anhydrase
Phosphorylase B
Catalase
Lactate
dehydrogenase
Total protein
range (ng)
ng protein/mmZ
Number of
points
measured
Slopeb)
Y-interceptb)
Correlation
coefficientb)
270
360
200
250
200
640
180
0.05 to 2.21
0.02 to 1.01
0.04 to 0.96
0.06 to 1.60
0.06 to 2.85
0.06 to 6.24
0.03 to 1.59
101 .I
231.5
165.8
83.3
26 .O
45.8
127 .O
9 .O
2.7
2.4
- 12.4
- 0.8
3.4
8.4
0.995
0.998
0.996
0.990
0.983
0.998
0.990
9.6 t o 240
0.06 to 1.57
107.5
4.4
0.990
5.4 to
7.2 t o
8.0 to
25.0 to
4.0 to
6.4 to
3.6 to
a) Proteins (Pharmacia purified molecular weight markers) were separated in a 10 % polyacrylamide gel and densities
and band areas determined with computerized microdensitometry. Some of the data in this table is illustrated in
Fig. 2.
b) Slopes, Y-intercepts and correlation coefficients were determined by linear regression analysis.
Simplified silver stain
Electrophoresis 1982,3, 17-23
21
Table 2. Ratios of slopes of staining: carbonic anhydrase 'lopea)
slope for other protein
Minutes
developed
Albumin
Ferritin
Catalase
Soybean
trypsin
inhibitor
Ovalbumin
25
15
9
0.268
0.25 3
0.254
0.231
0.185
0.3 24
0.336
0.310
0.334
0.534
0.250
0.243
0.203
0.209
0.198
0.184
0.200
0.156
0.127
0.141
0.102
0.110
0.112
0.177
a
6
-
Lactate
dehydrogenase
Phosphorylase B
0.246
0.236
0.240
0.245
0.315
1.403
1.753
1.093
1.1 14
1.303
a) Slopes for density versus protein concentration were determined for 8 purified proteins at 5 points during
image development in sodium carbonate/formaldehyde. Ratios were obtained by dividing the slope for
carbonic anhydrase by the slope for each of the other proteins at each time point. Gels were photographed
sequentially during image development and density measurements were made for 5 concentrations of each
protein at each time point.
can be performed on gels which were stained months previously as long as they were stored in an aqueous solution.
Studies utilizing [l4C1 proteins and autoradiography have
been used to demonstrate that silver stains detect most proteins in cell lysates [41. However, we have observed an acidic
protein in E. coli lysates which does not stain with the photochemical stain without recycling (Fig. 3). This acidic protein
may not permit the reduction of ionic to metallic silver in the
presence of dichromate, but it permits reduction to occur
when silver ions are present without dichromate, as in the
recycling procedure. Some silver stains are fairly specific for
particular proteins, perhaps because different mixtures of
metal ions detect subtle variations in protein structure. A
histological silver stain, containing copper, stains mainly
neurofilament polypeptides in gels of spinal cord homogenates [ 13].
The numerous image intensification methods which have
been developed for photography may also be used to intensify silver-stained electrophoretograms. Some modifications
in chemical concentrations and procedure timing are necessary to compensate for the variations in gel thickness and diffusion properties. This recycling procedure intensifies the image by the addition of silver; other metals such as copper,
mercury, chromium and uranium may also be used. Some intensification procedures increase image densities proportionally, while others, such as those employing copper iodide
and mercuric chloride, are superproportional. They increase
the image contrast by intensifying optically dense areas more
Table 4. Effects of recycling on protein spot and background densities
Cycle
Mean protein spota)
density
Mean background
density
1
2
3
10.2
20.2
25.6
0.4
0.6
1.1
a) Mean protein spot density was determined by measuring the densities of 20 E. coli proteins separated by two-dimensional electrophoresis (Fig. 3). Densities were calculated using a computer
program which finds the modal density in the vicinity of each
protein spot, subtracts this background from the average density
and multiplies this density by the spot area.
rapidly. Reagents such as uranium, mercuric iodide, and
chromium are subproportional in that they selectively intensify optically less dense regions.
Silver-stained gels may be destained by using photographic
destaining reagents or photographic reducers l131. These
destaining procedures also permit proportional, sub- and
superproportional effects. It is difficult to control the speed
or end-point of destaining procedures. Photographs should
be made of silver-stained electrophoretograms prior to and
during destaining procedures so that, if the desired end-point
is passed, the image can be preserved. Destaining can be
useful for clarification of the image in streaked or highly
stained regions of the gel (panel D, Fig. 3).
Table 3. Effect of varying amount of protein loading, with differential staininga)
3.4 Quenching effects of silver stains
Total
protein
Pg
Correlation coefficient obtained by measuring
densities of 45 protein spots per gelb)
10
25
50
100
~~
10
25
50
100
200
1.o
0.94
0.95
0.90
0.60
1.00
0.96
0.94
0.73
1.00
0.97
0.74
1.00
0.84
200
~
1.00
a) Differential staining was accomplished by varying the time of
image development in sodium carbonate/formaldehyde. An attempt was made to stain each gel to the same intensity.
b, Densities of 45 lymphocyte proteins were measured from twodimensional gels after differential staining with silver. Correlation
coefficients were computed using linear regression analysis.
Table 5 illustrates the effect of silver staining on the detection
of [l4C1 and 13Hl proteins by fluorography and autoradiography. This effect has been quantitated for two stains
developed in this laboratory [ 151. The histological silver stain
[3,41 almost completely quenches the detection of [3Hl proteins by fluorography. The photochemical silver stain (section 2.6), which uses less silver nitrate per gel, also causes
severe quenching of [3H] proteins, with only a faint appearance of a few major proteins. However, when silverstained gels containing 13H] proteins are destained with
photographic reducers before performing fluorography,
detection of the labeled proteins is partially restored for the
photochemical stain but not the histological stain. The photochemical silver stain has a negligible effect on autoradio-
22
Electrophoresis 1982, 3, 17-23
C. R. Merril, D. Goldrnan and M. L. Van Keuren
Table 5. Effect of silver staining on detection of [3H] and [14C]
protein+)
Staining method
Unstained
Coomassie Blue
Histological silver stain
Photochemical silver stain
Destained histological
silver stain
Destained photochemical
silver stain
Fluorography
of [ 3H] proteins
Autoradiography
of [' 4C] proteins
1oob)
1 oob)
85 * 3
N .D .c)
N.D.~)
93t 4
53t 3
982 6
N.D.~)
48f I
43 f 9
81 f10
stain is used, exposure of the gels to X-ray film for a longer
period of time is necessary. The use of the histological silver
stain is incompatible with fluorography of [3H] proteins.
However, one can use the photochemical silver stain for [3Hl
proteins by photographing the stained gel and then destaining it before fluorography. Spot densities on the X-ray film
will be quenched by approximately 50 %.
3.5 Color
graphic detection of [14C]proteins; the histochemical stain,
however, has a moderate quenching effect. This quenching
affects all protein densities within a gel by the same ratio; a
linear relationship between densities is therefore maintained
[ 151. For ''C-autoradiography, the photochemical silver
stain should not cause problems. If the histological silver
The photochemical silver stain described in section 2.6 is
fairly monochromatic. Spots appear brownish-black if image
development in sodium carbonate/formaldehyde is stopped
within 15 min. If image development is allowed to proceed
further, dense spots become saturated and color effects are
enhanced. With the histochemical stain, some liproproteins
were blue while glycoproteins were yellow, brown or red
[161. By modifying the silver stain, color effects can be enhanced [ 171. However, saturation and negative staining,
which usually occur with color enhancement procedures,
result in problems if protein quantitation is attempted on gels
which have been color-enhanced. Color may be useful in
identifying unknown proteins and in distinguishing overlapping spots 116, 171.
Figure 3. Image intensification of a two-dimensional gel containing E . coli
proteins (30 pg total protein). First (A) the gel was stained and described
(see section 2.6). Then (B) it was recycled through silver nitrate and
sodium carbonate/formaldehyde (see section 2.7) followed by a second
recycling (C). Background staining was reduced (D) by destaining (see
section 2.8) using a 10-fold dilution of the destaining solution. The arrow
in the intensified image in C, B and D indicates a protein which only stains
on recycling.
a) Mouse fibroblast proteins were separated on two-dimensional gels.
b) Mean percent densities f SE.M. of 13 proteins as compared to
densities obtained on unstained gels.
c, N.D. - not detectable on X-ray film due to quenching.
Electrophoresis 1982. 3, 17-23
3.6 Problems occurring with silver staining
Simplified silver stain
23
quantitative studies may be performed. Each protein has a
characteristic staining rate which may prove helpful in its
characterization. The silver stain may be modified to enhance color formation. However, caution must be exercised
if quantitative comparisons are required, since the enhancement of color may result in negative staining or saturation.
Despite the simplicity of the staining procedure, there are pitfalls which cause artifacts and lower sensitivity. Consideration should be given to the purity of the water and to the purity of the various reagents. Surface stain artifacts can be
reduced by handling gels carefully. The availability of
simplified silver-staining methods should encourage their use
in biochemical analyses.
The purity of the water is critical for maximal sensitivity.
Deionized water is required. Commercial formaldehyde may
vary in concentration from the required 37 %. Inadequate
fixation causes loss of sensitivity. Riboflavin and glycine
cause the gel to turn black. If the sodium carbonate/formaldehyde is not changed rapidly during initial image development (see sections 2.6 and 2.7) and agitated continuously, a precipitate forms which adheres to the surface of
the gel, causing a black discoloration. Pressure or exposure
to air or the presence of borate will also cause surface deposition. Usually, two horizontal lines are visible upon staining at
approximately 60000 and 67000 M,. These can also be
visualized, at times, by Coomassie Brilliant Blue R-250 stain- The authors acknowledge the meticulous technical asing. In two-dimensional gels in which SDS was used in sistance of Igor Prissovsky and Harry Kamerow and the exisoelectric focusing, there is an anodal region which does not pert manuscript preparation of Maxine Steyer.
stain. Touching gels, or permitting them to dry prior to staining, results in surface marks. Storage of gels in contact with Received October 13, 1981
each other, prior to complete fixation, can result in the
transfer of the proteins of one gel to the surface of an adja5 References
cent gel.
3.7 Staining of DNA in polyacrylamide gels
DNA fragments separated in 5 % polyacrylamide slab gels
can be stained using the photochemkal silver stain as
described. Gels should be washed free of buffer with distilled
water and need not necessarily be fixed. Optimal image
development Occurs Over 20-25 min in sodium carbonate/
formaldehyde. Placement of gels on a light box during this
step may result in some increase in Sensitivity. Sensitivity is
comparable to that observed with ethidium bromide using
fluorescent detection. Gels which have been stained with
ethidium bromide may subsequently be stained with silver.
4 Concluding remarks
The silver stain has been simplified; it now requires only three
solutions: dichromate, silver nitrate, and sodium carbonate/
formaldehyde. It has been optimized for rapidity, reproducibility and cost. The recycling procedure offers a simple
method for image intensification and allows detection of
previously unstained proteins. The sensitivity of this silver
stain is almost 200 times that obtained with Coomassie
Brilliant Blue R-250. As long as care is taken to keep the protein concentration of a spot or band less than 2 ng/mm2, and
images are not developed for longer than 15 min in sodium
carbonate/formaldehyde, saturation does not occur and
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