Pierce Silver Stain Kit for Mass Spectrometry

Thermo Scientific Pierce Silver Stain for Mass Spectrometry is a complete kit for rapid and sensitive silver staining of proteins in polyacrylamide gels and efficient destaining of excised gel pieces for mass spectrometry analysis.

This silver staining kit for mass spectrometry (MS) bundles components of the high-performance Pierce Silver Stain Kit with optimized reagents to destain spots for subsequent in-gel tryptic digestion to recover peptide fragments for proteomics analysis. The resulting MS-compatible product and protocol deliver outstanding sensitivity and maintains favorable conditions for the high-yield recovery and identification (sequence coverage) of proteins by mass spectrometry. Silver staining of 2D gels is now an important intermediate step in a set of procedures that leads ultimately to identification of specific proteins in the proteome by mass fingerprinting methods. The Pierce Silver Stain protocol provides peak staining performance, flexibility, reliability and robustness for applications such as MALDI-MS.

Highlights:

• Sensitive and fast staining – the low-background, easy-to-use silver stain provides subnanogram sensitivity, detecting proteins at less than 0.25ng per spot in 30 minutes after fixing
• Flexible staining protocol – fix gel for 15 to 30 minutes or overnight without any affect on staining performance; stain for 1 to 30 minutes (typically 2 to 3 minutes)
• Robust - effective silver stain for even difficult-to-stain basic proteins, such as lysozyme (pI 10) and chymotrypsinogen A (pI 9.2), which are detectable at 0.2ng and 0.5ng, respectively
• Simple, trouble-free spot preparation – stained spots in excised gel-pieces are easily destained and made ready for tryptic digestion in one hour
• MS compatible – reagents and protocol are optimized to provide excellent performance in mass spectrometry following extraction of peptides from stained 1D or 2D gels (SDS-PAGE)
• Complete kit – contains all reagents necessary for staining and destaining before in-gel proteolysis and peptide recovery for analysis by mass spectrometry
• Convenient – kit-components are stable at room temperature; saves valuable refrigerator space and eliminates the need to equilibrate reagents before use

Includes:

• Sensitizer, stain, developer and enhancer solutions for silver staining gels; Reagent solutions A and B for destaining excised gel pieces.

Product Details:

The Pierce Silver Stain Kit for Mass Spectrometry enables both first-time and experienced users to achieve consistent and reliable staining using high-, low- and gradient-percentage polyacrylamide gels in single-dimension and 2D formats. The optimized staining method ensures extremely sensitive staining while minimizing covalent crosslinking of protein to the gel matrix, which can inhibit protein-peptide recovery following in-gel proteolysis. The destaining reagents facilitate complete removal of silver from stained protein bands and maximum protein recovery for subsequent mass spectrometry analysis.

Silver staining methods generally use either glutaraldehyde or formaldehyde, which cause some covalent crosslinking of protein to each other and the gel matrix. To the extent that this crosslinking occurs, extraction or elution of protein from the gel will be inhibited. Pierce Silver Stain uses formaldehyde in the stain and developer working solutions. However, the procedure accompanying the Kit for Mass Spectrometry is optimized to maximize polypeptide recovery without greatly sacrificing sensitivity.

Staining protocol for the Thermo Scientific Pierce Silver Stain Kit for Mass Spectrometry.

Spot excision and band destaining protocol summary. Place each gel piece in a 0.5mL microcentrifuge tube. Mix Destain Working Solution (DWS) at recommended volumes for Reagent A, B and water. Add 200µL of DWS to each gel piece, mix gently and incubate for 15 minutes. Repeat once. Preparation for in-gel digestion. Wash three times with 25mM ammonium bicarbonate in 50% acetonitrile for 10 minutes each. Proceed with in-gel tryptic digestion (e.g., Thermo Scientific In-Gel Tryptic Digestion Kit, Part No. 89871).

Sequence coverage comparison for different gel stains. BSA, ovalbumin, chymotrypsinogen A and myoglobin (50ng each) were loaded onto mini gels and separated by SDS-PAGE. After electrophoresis, the respective gels were stained with Pierce Silver Stain for Mass Spectrometry, an MS compatible stain silver stain from Supply X and GelCode Blue Stain Reagent. Bands were excised and destained, subjected to in-gel tryptic digestion (Part No. 89871) and prepared for analysis by MALDI/MS. Proteins were not reduced or alkylated before in-gel tryptic digestion. For all proteins the Pierce Silver Stain for Mass Spectrometry performed better than or equal to the alternative silver staining method and Thermo Scientific GelCode Blue Stain (Part No. 24590).
	Thermo Scientific Pierce Silver Stain for MS	Supplier X Silver Stain for MS	Thermo Scientific GelCode Blue Stain Reagent
BSA Ovalbumin Chymo-trypsinogen A Myoglobin	63 13 21 40 5 13 47 4 9 32 6 19	53 6 11 44 1 2 41 2 5 31 3 10	40 7 18 42 1 2 41 1 2 38 1 3

2D gel stained with the Thermo Scientific Pierce Silver Stain Kit for Mass Spectrometry. Proteins of a rat mitochondrial extract were separated by IEF (pH 5-8 gradient) and SDS-PAGE, then stained. Ten spots were identified that stained well in three identical gels that were stained with three different stains (see table below). These spots were picked for in-gel digestion and MS analysis.

Related Resources:

Gel Stains Comparison and Selection Guide
Review of protein gel staining
Review of sample prep for mass spectrometry

Related Products:

In-Gel Tryptic Digestion Kit
Pierce Trypsin, MS Grade
Pierce Silver Stain Kit (same stain without spot-destaining reagents)
Pierce Silver Stain Rescue Reagent