Silver stain procedure and kit for same

A silver stain procedure wherein a substance capable of binding silver is treated with a glutaraldehyde solution, an aqueous silver salt solution, a reducing solution and an aqueous carbonate or sulfate solution and kit useful in practicing same.

FIELD OF INVENTION 
The pesent invention involves a novel silver staining method and a kit 
useful in practicing said method. 
BACKGROUND OF INVENTION 
There are a number of known methods useful in staining proteins which 
utilize silver. For example, L. Kerenyi, et al., Clin. Chim. Acta 38, 
465-467 (1972) describes a method for demonstrating proteins in 
electrophoretic, immunoelectrophoretic and immunodiffusion preparations 
whereby the preparations are treated with potassium ferrocyanide which is 
transformed during development into silver ferrocyanide then into 
colloidal silver grains. The physical developer contains anhydrous sodium 
carbonate, ammonium nitrate, silver nitrate, tungstosilicic acid and 
formalin, and the protein in the preparations stain dark brown with a pale 
gray background. 
R. C. Switzer, et al., Anal. Biochem. 98, 231-237 (1979) and C. R. Merril, 
et al., Proc. Nat'l. Acad. Sci. U.S.A., 76, No. 9, 4335-4339 (1979) 
describe a silver stain technique for detecting proteins and peptides in 
polyacrylamide gel which is a modification of de Olmos' neural, 
cupric-silver stain. The procedure consists of ten steps and utilizes an 
aqueous solution of silver nitrate and cupric nitrate and involves 
treatment with a diammine solution which is known to sometimes form an 
explosive silver amide complex. The proteins stain as dark spots on a 
darkened background. 
B. A. Oakley, et al., Anal. Biochem. 105, 361-363 (1980) simplified the 
above procedure of Switzer, et al., by reducing the number of steps 
involved to six and also reducing the amount of silver required without 
diminishing the sensitivity of the technique. However, the manner in which 
the proteins stain was not changed, i.e., dark stain on a darkened 
background. 
A further modification of the Switzer, et al., procedure was made by R. C. 
Allen, Electrophoresis I, 32-37 (1980) who increased the sodium to 
ammonium ion ratio which resulted in increased silver deposition. 
C. R. Merrill, et al., Anal. Biochem. 110, 201-207 (1981) modified and 
simplified the above procedure of Kerenyi, et al., adapting it to 
acrylamide gels. 
D. Goldman, et al., Clin. Chem. 26, No. 9, 1317-1322 (1980) report that 
when using a procedure essentially the same as that of Merrill, et al., 
(PNAS, 1976) and Switzer, et al., (Anal. Biochem., 1979) proteins from 
samples of cerebral spinal fluid stained in shades of yellow, red and 
blue. 
C. R. Merrill, et al., Science 211, 1437-1438 (1981) describe a silver 
stain procedure for proteins separated by two dimensional gel 
electrophoresis which requires treatment with potassium dichromate and 
nitric acid prior to staining with silver nitrate followed by washing then 
immersion in an image developer containing formalin and sodium carbonate. 
There is no indication of color development with this stain procedure. 
Poehling and Neuhoff, Electrophoresis 1981, 2, 141-147, describe a silver 
stain suitable for acrylamide gels of 0.5 to 1 mm thickness which requires 
a pretreatment with glutardialdehyde under controlled temperatures prior 
to staining with a diamine solution. 
Marshall and Latner, Electrophoresis 1981, 2, 228-235, describe a silver 
stain method which requires a treatment with paraformaldehyde and sodium 
cacodylate prior to staining with a modified diamine solution wherein 
methylamine is substituted for ammonium hydroxide. Ochs, et al., 
Electrophoresis 1981, 2, 304-307, and Sammons and Adams, Electrophoresis 
1981, 2, 135-145, describe a silver stain procedure of which the present 
invention is a modification. 
With the exception of the 1980 Goldman, et al., procedure and the method of 
Sammons and Adams all of the silver stain techniques known heretofore only 
stain proteins in varying shades of brown or black. 
The present invention provides a unique stain procedure which not only is 
highly sensitive, but also, enables one to stain a variety of substances 
including proteins in varying shades of color. 
SUMMARY OF INVENTION 
The present invention is a method for staining various types or classes of 
compounds which is highly sensitive enabling one to detect picogram 
quantities of compounds or substances, and also is unique in that the 
method imparts color to the various substances staining them in varying 
shades of blue, green, red, and yellow. The present method will stain any 
substance that is capable of binding silver. 
The present invention provides a novel method for staining substances 
capable of binding silver and which are supported in or on a matrix which 
comprises the steps of oxidizing said substance with a glutaraldehyde 
solution, washing with water, equilibrating said substance in a suitable 
silver salt solution, then subjecting said substance to a reducing 
solution, and immersing said substance in a first, then a second and third 
sulfate or carbonate salt solution. 
The present method is particularly suited for staining proteins. The 
present method is particularly useful in enhacing stain sensitivity in 
ultrathin gel preparation. The color can be achieved by prolonging the 
reduction step and adds a new dimension to the separation and 
identification of proteins, as well as other substances, by 
electrophoresis by aiding for example in the resolution of overlapping 
proteins and identifying members of a family of proteins by their 
characteristic color. Thus the method of the present invention which is 
both sensitive and reproducible will facilitate the identification of 
substances or compounds that are diagnostic markers and/or primary lesions 
of most genetic diseases including the major diseases such as diabetes, 
atherosclerosis and cancer. In addition, pathological changes in tissues 
can be correlated with changes in certain substances, particularly 
proteins. The present invention provides a means to enhance ones 
understanding of normal and disease conditions. Also, the present method 
is particularly useful in detecting minute amounts of proteins separated 
from samples required by forensic medicine. 
Another aspect of the present invention is a kit wherein the component 
parts are assembled in a manner to facilitate the practice of the silver 
staining method of the present invention, said kit comprising multiple 
containers having therein appropriate amounts of reagents necessary to 
practice said method as follows: 
(a) a container having therein a solution of 2.5-25% glutaraldehyde; 
(b) a container having therein a suitable silver salt; 
(c) a container having therein an appropriate base solution; 
(d) a container having therein an aldehyde solution; and 
(e) a container having therein an appropriate amount of a carbonate or 
sulfate salt. 
Generally the quantity of oxidizing reagent contained in the kit for 10 
gels (8 cm.times.7 cm.times.0.1 mm) will be: 60 ml of a solution of 25% 
glutaraldehyde; the quantity of silver salt contained in the kit will be 
up to 0.6 grams and preferably will be 0.4 to 1.0 grams; the quantity of 
base will vary from 100 to 150 ml; the quantity of aldehyde will vary from 
about 0.50 to 2.0 ml; and the amount of carbonate or sulfate salt will 
vary from about 0.5 to 2.0 grams. The glutaraldehyde solution is buffered 
with 1 to 2% of sodium cacodylate, pH 7.3.

DETAILED DESCRIPTION OF INVENTION 
The method of the present invention is useful to stain any substance or 
compound which is capable of binding silver. Such substances include 
proteins, polypeptides, amino acids, nucleic acids and polymers thereof, 
lipids, carbohydrates including starches and sugars of various classes, 
e.g., oligosaccharides, or polysaccharides, such as mucoitin sulfate, 
lipoproteins, glucoproteins, nucleoproteins, including ribonucleic- and 
deoxyribonucleic-protein complexes, mucopolysaccharides, e.g., 
chondroitin, proteoglycans, mucolipids, such as ganglioside, mucoproteins, 
and glycolipids. We have found that the present method is particularly 
useful in identifying families of proteins, i.e., proteins comprised of 
the same protein subunits but which may be conjugated to other chemical 
entities, such as, a saccharide thereby altering the overall net charge or 
character of the protein. 
It is important when staining the sample of substance supported in a 
matrix, such substance so supported being referred to hereinafter as 
"matrix preparation," by the method of the present invention that said 
matrix preparation be washed thoroughly to remove any components which may 
interfere with the uptake of silver by the substance to be stained. The 
matrix preparation should be washed repeatedly, for example, in a lower 
alkanol, such as methanol or ethanol containing a mild acid, such as, 
trichloroacetic acid or acetic acid. Generally, for a 0.1 mm thick gel, 
washing for 1 to 3 hours is adequate with several changes of solution 
using for each milliliter (ml) of matrix 15 ml of solution per change. Of 
course, less time is required for thinner gels. This washing procedure is 
commonly referred to as fixation/washing. 
The present novel staining method can be used to stain virtually any 
substance which is capable of binding silver and typically is useful in 
staining such substances which have been separated using one or two 
dimensional electrophoresis techniques. The matrix in which the substance 
to be stained is supported can be comprised of any materials which are 
commonly used in electrophoretic procedures. For example, the matrix may 
consist of derivatized paper that is useful for electrophoretic protein 
transfer, cellulose acetate, starch gel, agarose, Sephadex beads, or 
polyacrylamide, and may vary in thickness from an ultrathin matrix of 
about 0.05 mm to 0.5 mm. It may be desirable to attach the matrix on a 
nylon mesh, a glass plate, or a plastic sheet for additional support. As 
is known from standard electrophoretic techniques, the density and pore 
size of the matrix may also vary. A preferred matrix composition is 
polyacrylamide, varying from 0.05 mm to 0.5 mm in thickness with 
80-100.mu. being the most preferred thickness. 
We have found that the present method is especially useful for staining 
substances, and, in particular, proteins that have been separated by two 
dimensional electrophoresis on polyacrylamide gels. The method of the 
present invention is particularly useful for staining substances separated 
on ultrathin gels prepared by the method of Gorg, et al., Anal. Biochem, 
1979, 89, 60-70, and Radola, Electrophoresis, 1980, 1, 43-56. 
With the method of the present invention we have been able to detect 
picogram quantities of substances and obtain matrix preparations which 
stain in varying shades of blue, green, yellow and red. The background of 
the matrix preparation stains in light shades of yellow to orange upon 
which the brightly colored substances are detected easily. The intensity 
of the color will vary somewhat with the thickness of the matrix as well 
as with the concentration of the substance to be stained. For best results 
a matrix of 0.5 mm thickness of 10% to 20% polyacrylamide gel is used. 
The novel silver stain procedure described and claimed herein is highly 
sensitive, gives high resolution of substances, is reproducible, is easy 
to use, and is efficient in that matrix preparations can be stained 
batch-wise. Additionally the entire procedure is carried out at room 
temperature, i.e., about 18.degree. to 27.degree. C. with standard room 
lighting, there being no requirement of elevated temperatures or special 
light control. 
The oxidizing solution consists of having a concentration of from 2.5 to 
20% glutaraldehyde. Generally 3 to 15 ml per gel is used and 1 to 2% (w/v) 
sodium cacodylate, pH 7.3. Following treatment with glutaraldehyde it is 
important that the matrix preparation be thoroughly washed with distilled 
water. Washing for one to 6 hours with changes every 15 to 20 minutes is 
suitable. 
Suitable silver salts which may be used in the present invention are those 
which will dissociate in water to give free silver ion and which will not 
complex with the reducing agents employed in the method. Aqueous solutions 
of silver nitrate or silver acetate are particularly suitable for use in 
the present invention. The silver salt is dissolved in water, generally 
distilled water, at a concentration of from about 1 to 6 grams of silver 
salt per liter of water. The lower concentration of silver salt, i.e., 1 
gram/liter results in reduced sensitivity and intensity and the background 
of the matrix strains lighter than at the higher concentrations of silver 
salt. Concentrations of silver salt varying from about 3 to 6 grams of 
salt per liter of water are preferred. The matrix preparation is 
equilibrated in the silver salt solution for a minimum of about 10 minutes 
using for each 1 ml of matrix about 15 ml of solution. Of course the 
thicker matrix preparations require more time to equilibrate than do the 
thinner matrix preparations. 
Following equilibration in the silver salt solution the matrix preparation 
is subjected to a reducing solution. The reducing solution may be sprayed 
onto the matrix preparation or the matrix preparation may be immersed in 
the reducing solution. The reducing solution consists of a base such as 
aqueous potassium hydroxide or sodium hydroxide and an aldehyde, such as, 
formaldehyde, acetaldehyde, n-butyraldehyde, or glutaraldehyde. The 
concentration of base may vary from about 0.5 to 1.0 N with 0.7 N base, 
preferably sodium hydroxide, being the most suitable. The preferred 
reducing agent is formaldehyde and generally a 37% solution is employed. 
Generally the reducing solution will comprise about 0.75 to 1.5 ml of 37% 
formalin per about each 150 ml of aqueous base. A preferred reducing 
solution consists of per each 150 ml of 0.75 N NaOH, 1.1 ml of 37% 
formalin. For each ml of matrix preparation one should use about 15 ml of 
reducing solution. 
It is recommended that in preparing the reducing solution as well as other 
aqueous solutions described herein one use distilled deionized and 
degassed water. The reduction generally requires about 30 seconds to 5 
minutes, and when the matrix preparation is submersed in the reducing 
solution agitation at about 40 cycles per minute is recommended. The time 
required for reduction varies with the thickness of the gels with time 
required increasing as the matrix thickness increases. The reaction time 
could be shortened if the temperature is raised. Generally the components 
of the reducing solution are mixed together and the matrix preparation 
previously equilibrated in the silver salt solution is brought into 
contact with the resultant solution. 
Following reduction the matrix preparation is immersed for about one to 5 
minutes, preferably one minute, in three sequential solutions of 0.075 to 
1.5%, preferably 0.75%, anhydrous sodium or potassium carbonate or sodium 
or potassium sulfate having a pH of 11 or greater. The matrix preparation 
should then be rinsed with water and air dried. Sodium carbonate is 
preferred for this step. The stained preparation may be photographed by 
standard well known techniques. 
As indicated hereinabove standard electrophoretic procedures may be used to 
separate the substances to be stained in either one dimension or two 
dimensions. When such substances have been separated using standard two 
dimensional electrophoresis on polyacrylamide gel, it is very important to 
remove the SDS and electrophoretic buffer salts during the 
fixation/washing step. Also it is recommended that reagents and water be 
of highest quality. 
When preparing the sample of substance to be stained the concentration for 
each sample will have to be adjusted individually. Generally, we find that 
samples should be diluted 10 to 50 times of those used for staining by 
conventional Coomassie Blue procedures. 
A preferred general procedure is the following. 
GENERAL PROCEDURE 
Tissue samples or samples of substances to be stained are prepared and run 
on first dimension gels according to the procedures of Gorg, et al., 
ibid., and Radola, ibid., and second dimension gels according to the 
procedure described by Anderson and Anderson Anal. Biochem. 85, 331-354 
(1978). Second dimension gels are fixed in 50% ethanol/10% acetic acid for 
two or more hours with one change of fixative. The gels are then washed 
two times in 25% ethanol/10% acetic acid and 6 times in water with changes 
every 30 minutes to remove any interfering substances. The gels are then 
subjected to an oxidizing solution of 5-10% glutaraldehyde for up to 12 
hours then washed thoroughly for one to 6 hours with changes every 15 to 
20 minutes with distilled water. Next the gels are soaked for about 10 
minutes to one hour in 4 g/l AgNO.sub.3 in water. Gels are removed from 
the silver solution and processed singly or in groups of up to 10 or more. 
Single gels are then placed in a rectangular glass dish 
10.times.10.times.1 cm. To this dish is added reducing solution which 
consists of 15 ml 0.75 N NaOH and 0.1 ml of 37% formaldehyde. The dish is 
placed on a shaker for 30 seconds, then the reducing solution is poured 
off and replaced with an equal volume of 0.75% Na.sub.2 CO.sub.3. The 
sodium carbonate solution is changed 3 times at one minute intervals. For 
larger gels, proportionately larger volumes of reducing solution and 
Na.sub.2 CO.sub.3 solution and longer treatment times are used. 
EXAMPLE 1 
Human granulocytes (10.sup.7) were solubilized in one ml of 1% Triton 
X-100. A lysate was prepared by centrifugation 100,000.times.g for one 
hour. A one mm.sup.2 square piece of Whatman No. 2 filter paper was 
saturated with the above protein extract. The filter paper was then placed 
on an ultrathin gel, 7.times.8 cm.times.100.mu. thickness and subjected to 
isoelectric focusing according to the procedure of Allen, Electrophoresis, 
1980, Vol. 1, p. 32. The gel was fixed in 12.5% trichloroacetic acid and 
dried at 60.degree. C. Prior to staining the gel was soaked in distilled 
water for one hour then placed in 25 ml of 3% glutaraldehyde overnight. 
The gel was then washed with five changes of distilled water (100 ml per 
change) for a 6 hour period. The gel was equilibrated in 15 ml of silver 
nitrate (4 g/l) for 20 minutes. The gel was then placed in a reducing 
solution comprising 15 ml of 0.75 N sodium hydroxide and 0.1 ml of 37% 
formaldehyde for 30 seconds with gentle agitation. The reducing solution 
was then poured off and replaced with 15 ml of 0.75% sodium carbonate for 
one minute followed by two equal volume changes of 0.75% sodium carbonate 
solution at one minute intervals. The gel was then removed, rinsed with 
distilled water and air dried. The separated proteins appeared as brown to 
black bands on a light amber stained background. 
When in the above procedure the gel is left in the reducing solution for 5 
minutes the protein bands stain shades of color varying from red, yellow 
and blue, however it appeared that the sensitivity was decreased. 
EXAMPLE 2 
The significant reagents for the performance of the staining procedure are 
assembled into a mercantile kit, specifically, a kit for marketing to 
qualified individuals to perform the staining procedure. The kit comprises 
as basic components at least four containers, one of each having therein 
appropriate amounts of a glutaraldehyde solution; a suitable silver salt; 
a suitable base; a suitable aldehyde reducing agent; or an appropriate 
amount of sodium carbonate. 
A preferred kit composition is one designed to stain 10 gels which are 
7.times.8 cm and 0.1 mm thick wherein one container has therein 60 ml of 
25% glutaraldehyde; one container has therein 0.6 grams of silver nitrate; 
another container has therein 9 ml of 50% sodium hydroxide; another 
container has therein 1.2 ml of 37% formalin; and one additional container 
has therein 3.4 grams of sodium carbonate.