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SilverXpress® and SilverQuestTM Silver Staining Kits
TABLE OF CONTENTS
PRODUCT DESCRIPTION
SHIPPING CONDITIONS
STORAGE CONDITIONS
STABILITY
QC SPECIFICATIONS
PROTOCOL & APPLICATION NOTES
Guidelines for SilverXpress® Silver Staining Kit
Guidelines for SilverQuestâ„? Silver Staining Kit
ALTERNATE PRODUCTS & COMPATIBILITY
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBER
COMPONENTS
ASSOCIATED PRODUCTS
RELATED TECHNICAL SUPPORT NOTES
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PRODUCT DESCRIPTION
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SilverXpress® Silver Staining Kit:
The basic mechanism of silver staining is the reduction of silver nitrate to metallic silver at a protein band. The bands are
visualized where the silver grain deposits.
There are two primary types of silver staining processes; chemical development and photo development.
The chemical development stains utilize ammonium hydroxide to form silver diamine complexes; the bands are
visualized by acidification.
The photo development stains utilize light to reduce silver ions to metallic silver; this type of stain is less sensitive
than the chemical development silver diamine stains.
The SilverQuest� Silver Staining Kit is a chemical development silver stain and as such is highly sensitive. SilverXpress®
provides nanogram-level sensitivity with minimal background in a little over one hour. The procedure actually takes less total
time than is required for standard Coomassie staining.
SilverQuestâ„? Silver Staining Kit:
The SilverQuestâ„? Silver Staining Kit provides a rapid and easy method to silver stain proteins in polyacrylamide gels. Silver
staining allows detection of most proteins, with 30-fold higher sensitivity than colloidal Coomassie® G-250 staining. This kit
is specifically designed to provide sensitive silver staining compatible with mass spectrometry analysis. This kit also
addresses staining for low levels of protein prior to sequencing/mass spectrometry analysis.
SHIPPING CONDITIONS
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The SilverXpress® Silver Staining Kit shipped at room temp or blue ice.
The SilverQuestâ„? Silver Staining Kit is shipped at room temperature.
STORAGE CONDITIONS
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Store the SilverXpress® Silver Staining Kit at 4°C.
Store the SilverQuestâ„? Silver Staining Kit at room temperature.
STABILITY
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The SilverXpress® Silver Staining Kit is stable for 6 months at 4°C. The Sensitizer, Stainer A and Stainer B reagents should
be used under a hood. See MSDS.
The SilverQuestâ„? Silver Staining Kit is stable for six months when stored at room temperature.
QC SPECIFICATIONS
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SilverXpress® Silver Staining Kit
Different dilutions of Mark 12. Unstained Standard are electrophoresed on a 1.0 mm, Novex ®Tris-Glycine and Tricine gels.
The gel is stained with SilverXpress® Silver Staining Kit as described in the product manual. Functional criteria are:
Silver staining must detect at least 1 ng protein.
At 1:10 dilution, seven protein bands of the Mark 12.
Unstained Standard must be visible on a 12% and 4-20%.
Novex® Tris-Glycine gel and eight protein bands on the 10- 20% Novex® Tricine gel.
Background must be free of dark blotches, uneven staining, and contaminant bands.
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SilverQuestâ„? Silver Staining Kit
Invitrogen qualifies the SilverQuest. TM Silver Staining Kit using the Basic Staining Protocol listed in the manual. Different
dilutions of BSA (0.3-2.5 ng) in 1X NuPAGE® LDS Sample Buffer are electrophoresed on a 1.0 mm, NuPAGE® Novex® 4-
12% Bis-Tris Gel using NuPAGE® MES-SDS Running Buffer. After staining, the gel is destained using the Destaining
Protocol on page 14. Functional Criteria are:
Silver staining must detect 0.3 ng BSA.
Background must be light and free from dark spots, uneven staining, or contaminant bands.
Destaining solutions must completely destain the protein bands within 15 minutes.
PROTOCOL AND APPLICATION NOTES
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Guidelines for SilverXpress® Silver Staining Kit
Standards
Sample loading
Staining Containers
Shaker
Water
Solutions
Detection
Nucleic acid staining (DNA and RNA)
Notes the SilverXpress® Staining Kit
Guidelines for SilverQuestâ„? Silver Staining Kit
For optimal staining results, follow these guidelines
Samples Containing High Concentration of DTT
Comments on procedures
Mass Spectrometry applications
Notes for the SilverQuestâ„? Staining Kit
Guidelines for SilverXpress® Silver Staining Kit
Standards:
Mark12 Wide-Range Standard is recommended for Tris-Glycine, Tris-Tricine and NuPAGE® gels; dilute 1:20 and load
5µl/lane.
Sample loading:
For optimal performance, prepare 1x running buffer in ultra pure water and use the 1x running buffer to rinse gel wells 4
times before sample loading.
Staining Containers:
Be sure to use clean round containers and designate these containers for silver staining purposes only.
Make sure that the container diameter and depth is sufficient to permit gel coverage with 100ml of solution per gel.
A container which measures 14cm in diameter and 5.5cm in height is ideal.
The StainEase Gel Staining Tray is a 14.5cm diameter tray with a clear polycarbonate outer container and lid and a
removable white polycarbonate strainer insert. It is ideal for preventing breakage during the multiple solution
changes and for easily visualizing band development.
Shaker:
Set at 1 revolution per second.
Water:
Always use ultra pure water (18 MEGOHM/cm resistance recommended) for solution preps and gel rinses,
container washing and running buffer preparation.
Water of less than 18 MEGOHM/cm may increase the gel background and/or impair band development.
Store ultra pure water in glass containers. Water stored in plastic containers can become contaminated.
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Solutions:
Avoid cross contamination of kit reagents; especially between Stainer A and Stainer B.
The order of adding A, B and ultra pure water is not important.
Use disposable pipettes to reduce cross contamination.
Make all solutions fresh (should not be older than 24hr).
Detection:
Detection is in the nanogram levels. As little as 0.86 ng of BSA can be detected on a 1.0mm Novex® 4-20% Tris-
Glycine gel.
Samples reduced with dithiothreitol (DTT) will be less sensitive than non-reduced samples. To obtain optimal
sensitivity with DTT reduced samples, use the Tris-Tricine protocol.
Non-reduced samples will have a slightly higher background than reduced samples. This will not affect detection
limits.
Silver staining sensitivity often depends on the specific protein structure. If dealing with a protein that is difficult to
silver stain, Coomassie staining prior to silver staining may reduce the protein-to-protein variability and may result
in higher sensitivity. If you are using SilverXpress® to stain a gel that has been Coomassie Stained, omit the Fixing
step in the SilverXpress® protocol and proceed directly to the Sensitizing step.
Nucleic acid staining (DNA and RNA)
Skip the fixation step and change the composition of the sensitizer solution to 2 ml of sensitizer and 198 ml of ultra-
pure water. This modification provides 0.3 ng sensitivity down to 50 bases, whereas the normal protocol provides
0.9 ng sensitivity down to 50 bases and ethidium bromide's sensitivity is approximately 10 ng down to 50 bases. To
increase sensitivity, use 7.5 ml of Stainer A instead of 5 ml or eliminate the 2nd wash step (Note: this may also
increase background).
Notes for SilverXpress® Silver Staining Kit
The SilverQuestTM and SilverXpress® Silver Staining Kits have very comparable sensitivity levels. The advantage
of the SilverXpress® kit is the ability to obtain a crystal clear background while the SilverQuestTM offers both a 30
minute microwave procedure and the mass spectrometry compatibility.
The SilverXpress® Staining kit can be used to stain samples loaded and run with β-mercaptoethanol but you may see
decreased sensitivity. The results can be improved by using the Tricine gel protocol, which incorporates longer stain
and wash times.
The SilverXpress® Staining Kit should be fine to use if it is accidentally stored in the freezer.
Glutamic Acid, Aspartic Acid, Cysteine Thiols are the amino acids most reactive with the silver stain.
The sensitizing step is the optimal point to stop the procedure. Although some other kits recommend leaving gels in
the fix step, we have found that overnight fixation diminishes stain performances.
The following is a list of visual cues which can serve as landmarks of a properly completed step within the
SilverXpress® Kit. These cues, as well as closely adhering to the protocol appropriate for your gel and sample type,
will help ensure that SilverXpress® Kit will yield the best results possible.
The stack of Novex® mini-gels or any gel cast with a low percentage stacking gel displays a whitish,
opaque appearance in comparison to the separating gel after the sensitizing step is carried out. This is a
good indicator that the sensitizing step was performed correctly.
Mini-gels curl up into a cylinder and float on the surface during the first water wash after the sensitizing
step. This is normal and will not affect staining performance.
When Stainer A and Stainer B are added together, there should be a brown precipitate formed, which is
visible only momentarily. This brown “flash� is a good indicator that the staining solutions have been
mixed correctly. In the event that the brown color does not revert to clear, discard the solutions, obtain
clean glassware and remix. Also, be careful not to cross contaminate the Stainer A and B bottles.
For silver staining of NuPAGE® gels using SilverXpress®, use gels that have been stored at 4°C should be used to
reduce background levels. Staining may be further improved by using a SilverQuestTM staining kit.
An alternative fixer used is 12.5% TCA, 4% 5-sulfosalicylate. Fix with this solution for 30 minutes, followed by a
30-minute wash in deionized water, and continue the stain protocol. This may result in slightly higher background
relative to the normal fixative procedure.
Guidelines for SilverQuestâ„? Silver Staining Kit
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For optimal staining results, follow these guidelines:
Wear rubber gloves that have been rinsed with deionized water while handling gels.
Use clean containers and designate these containers for silver staining purposes only.
The size of the container should permit free movement of the gel during shaking and complete immersion in
solution while staining.
The gels should not be handled with bare hands or metal objects and avoid applying pressure on gels while handling
or changing solutions.
Teflon coated stir bars and clean glass containers should be used to prepare reagents.
Cross contamination of kit reagents should be avoided.
Use freshly made solutions.
Samples Containing High Concentration of DTT:
If your sample contains high concentrations of DTT (>50 mM), silver staining may result in streaking and yellow
background. To avoid streaking, perform reduction and alkylation of the sample as described below:
Reduce your sample with freshly prepared DTT to a final concentration of 17 mM and heat the sample at
70°C for 10 minutes.
Alkylate the sample with freshly prepared iodoacetamide to a final concentration of 35 mM and heat the sample at
70°C for 10 minutes.
Add SDS sample buffer without DTT to the reduced and alkylated sample.
Proceed for electrophoresis and perform silver staining as described in the SilverQuestâ„? Silver Staining Kit manual.
Comments on procedures:
Choose the procedure appropriate for your type of gel and sample.
All solutions should be prepared according to the chart preceding the protocol. For best results, solutions should be
made fresh prior to beginning the staining process. The Fixing, Sensitizing and Staining Solutions should be made
under a hood. Cap solutions after use to minimize their exposure to external environment.
The volume of all solutions and time of incubations, including water washes, should be exact. Any deviations from
the protocol may result in poor band development and/or high background.
Mass Spectrometry applications
Silver staining of proteins followed by mass spectrometry analysis is a sensitive technique for protein identification in
the field of proteomics. In this method, proteins are separated by 2-D gel electrophoresis and silver stained. Since
extraction of proteins from the gel is difficult, in-gel digestion of proteins with proteases (mostly trypsin) is used to
generate peptide fragments. These peptide fragments are analyzed on Matrix Assisted Laser Desorption Ionization Mass
Spectrometry (MALDI/MS) to determine their exact mass. Resulting peptide masses are then subjected to various
database searches to identify the protein by comparing the peptide masses.
The SilverQuestâ„? Silver Staining Kit is specifically modified to be compatible with mass spectrometry analysis as
follows:
The sensitizing solution does not contain glutaraldehyde.
For optimal mass spectrometry analysis, it is important to obtain complete trypsin digestion of the protein sample.
Most silver staining kits contain glutaraldehyde-based sensitizers. Glutaraldehyde modifies lysine residues and
prevents complete trypsin digestion (Rabilloud, 1990). Glutaraldehyde also reduces the efficiency of protein
extraction from the gel by cross linking two lysine residues (Rabilloud, 1990). Since the SilverQuestâ„? Silver
Staining Kit does not include a glutaraldehyde-based sensitizer, trypsin digestion and extraction of peptides from the
gel is greatly improved.
The SilverQuestâ„? Silver Staining Kit includes destaining solutions that effectively remove silver ions from protein
bands in polyacrylamide gels (Gharahdaghi et al., 1999). This improves trypsin digestion and subsequent mass
spectrometry coverage of the protein, as silver ions are known to inhibit trypsin digestion of proteins (Chambers et
al., 1974).
Trypsin digestion and the preparation of samples for mass spectrometry are discussed in detail in the manual. See
manual for suggestions for MALDI MS analysis.
Notes for SilverQuestâ„? Staining Kit
It is always recommended to destain prior to MS, no matter what machine is used for MS analysis (i.e. MALDI/MS,
LC/MS). Destaining ensures no adducts of the stain will be present as a contaminant. Also, if you don't destain, the
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stain will denature the trypsin. As far as the thickness of gel to use, the 1mm is always better. The enzyme can get
into the gel easier.
TCA has never been tested in the extraction protocol. Therefore, we do not recommend using it instead of TFA. If
the researcher does not have TFA, use 100mM acetic acid instead, but the extraction efficiency may suffer for it.
Theoretically, the yield between Coomassie and SilverQuestâ„? sequencing should be the same but it is not
comparable between the two systems. The yields should be typically lower with silver quest because the amount of
protein used is less. However, if using the same amounts of protein and stained the yield will give similar results
with subsequent sequencing/mass spectrometry analysis.
Both SilverXpress® and SilverQuest� can stain peptides. SilverXpress® works well on the Insulin B-chain
(3.5kDa), but not on the A-Chain (2.5kDa, 21aa/4Cys). SilverQuestâ„? works very well on both chains and is the
only silver stain that been shown to stain the A-Chain well, so is probably the best to recommend.
It is possible to transfer the protein samples to a membrane after completely destaining the SilverQuestâ„? gel, due
to the lack of glutaraldehyde in the sensitizing solution.
After staining is complete, gels can be stored in water O/N @ 4°C and used later for MS analysis. Some protocols
call for storage in 1% Acetic Acid and this is also acceptable. Another option is to cut out the band of interest and
store the slice in water @ 4°C. For more specific requirements, customers should check with their core Mass. Spec.
facility.
PRODUCT DOCUMENTATION
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Brochures Citations Cell lines
COA FAQ Licensing
Manuals MSDS Newsletters
Vector Data
REFERENCES
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1. Chambers, J. L., Christoph, G. G., Krieger, M., Kay, L., and Sroud, R. M. (1974). Silver Ion Inhibition of Serine
Proteases. Biochemical and Biophysical Research Communications 59, 70-74.
2. Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., and Mische, S. M. (1999). Mass Spectrometric
Identification of Proteins from Silver-Stained Polyacrylamide gel: A method for the Removal of Silver Ions to
Enhance sensitivity. Electrophoresis 20, 601-605.
3. Rabilloud, T. (1990). Mechanisms of Protein Silver Staining in Polyacrylamide gels: A 10-year Synthesis.
Electrophoresis 11, 785-794.
PRODUCT NAME & CATALOG NUMBER
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Name Size Part Number Catalog Number
SilverXpress® Silver Staining Kit 1 kit (Reagents to satin 25 gels) LC6100 LC6100
SilverQuestTM Silver Staining Kit 1 kit (Reagents to stain 25 gels) 451001 LC6070
COMPONENTS
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SilverXpress® Silver Staining Kit
Name Size Part Number Catalog Number Information
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Sensitizer 125ml 467012 (LC7012) Contains Glutaraldehyde
Stainer A 125ml 467010 LC7010 Contains Silver Nitrate
Stainer B 125ml 467011 LC7011 Contains Ammonium Hydroxide & Sodium Hydroxide
Developer 125ml 467013 Contains Formaldehyde & Citric Acid
Stopper 125ml 467014 LC7014 Contains Citric Acid
Sufficient reagents are supplied to stain 25 mini-gels
SilverQuestâ„? Silver Staining Kit:
Component Size Part Number Color
Sensitizer 250 ml 461161 Orange
Stainer 25 ml 461162 Clear
Developer 250 ml 461163 Pink
Developer Enhancer 2 ml 461164 Clear
Stopper 250 ml 461165 Clear
Destainer A 60 ml 461166 Yellow
Destainer B 60 ml 461167 Clear
Sufficient reagents are supplied to stain 25 mini-gels.
Fixing solution if needed (not supplied): (40 % ethanol, 10% acetic acid, made in Ultra pure water).
ASSOCIATED PRODUCTS
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NuPAGE® Novex® 4-12% Bis-Tris Gels Cat. # NP0321BOX
Novex® 10% Tris-Glycine Gels Cat. # EC6075BOX
SilverXpress® Silver Staining Kit Cat. # LC6100
XCell SureLock. Mini-Cell Cat. # EI0001
Mark 12. Unstained Standard Cat. # LC5677
BenchMark. Protein Ladder Cat. # 10747-012
DryEase® Mini-Gel Drying System Cat. # NI2387
StainEase® Staining Tray Cat. # NI2400
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