Silver stain optimized for mass spectrometry

The Thermo Scientific Pierce Silver Stain for Mass Spectrometry Kit bundles the high-performance Pierce Silver Stain II with an efficient gel-destaining chemistry and an optimized protocol. The resulting mass spec compatible product delivers outstanding sensitivity and maintains favorable conditions for the recovery and identification of protein by MS.

Silver staining of 2-D 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 kit performance, flexibility, reliability and robustness in MS-targeted applications.

• Sensitivity - this low-background, easy-to-use silver stain provides sub-nanogram sensitivity, detecting less than 0.25 ng protein/spot in 30 minutes after fixing; spots are de-stained and ready for tryptic digestion in one hour
• MS compatibility - provides excellent MS performance on 1-D and 2-D gels; MALDI-MS results are superior to other MS-compatible stains
• Complete and ready-to-use - turnkey kit contains all reagents for staining and destaining process before MS analysis; contains sufficient destain reagents for 500 excised spots
• Flexibility - fix in 15-30 minutes or, for convenience, overnight; stain in 1-30 minutes (typically 2-3 minutes)
• Robust - effective for difficult-to-stain basic proteins such as lysozyme (pI 10) and chymotrypsinogen A (pI 9.2), detectable at 0.2 ng and 0.5 ng, respectively
• Convenience - room temperature-stable kit components eliminate the need to occupy valuable refrigerator space

SilverSNAP Stain for Mass Spectrometry Staining and Destaining Protocol after Fixing the Gel
1. Wash gel 2X with 10% EtOH Follow by 2X water wash (5 min. each wash)	2. Sensitize gel Mix 1:500 Sensitizer:Water Sensitize gel for 1 min. Follow by 2X water wash (1 min. each wash)	3. Silver stain Mix 1:100 Enhancer:Stain Incubate for 5 min. Follow by water wash
4. Develop gel Mix 1:100 Enhancer:Developer Wash 2X with water (20sec./wash) Add developer; incubate 2-3 min.	5. Stain development stop Add 5% HAc; incubate 10 min. Water wash gel Gel is ready for spot excision and destaining
6. Destain excised spots Place each gel piece in a 0.5 ml microcentrifuge tube Mix Destain Working Solution (DWS) at recommended volumes for Reagent A, B and water a ) Add 200 µl of DWS to each gel piece b) Mix gently - incubate 15 min. (Repeat a. and b.above)
7. Prep for In-gel Digestion Wash 3X with 25mM NH4HCO3:50%AcCN (10 min. each wash) Proceed with in-gel tryptic digestion

	Pierce Silver Stain for MS	Competitor I MS Stain	GelCode Blue Stain Reagent
Protein 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
Sequence Coverage Comparison. 50 ng each of BSA, Ovalbumin, Chymotrypsinogen A and Myoglobin preparations were loaded onto separate SDS-PAGE gels. After electrophoresis, the respective gels were stained with Pierce Silver Stain for Mass Spectrometry, a competing MS compatible stain and GelCode Blue Stain Reagent. The resultant bands were excised and destained, subjected to in-gel tryptic digestion (Product #89871) and prepared for analysis by MALDI/MS. Proteins were not reduced or alkylated prior to in-gel tryptic digestion. The results of the analysis are summarized above. In all cases the Pierce Silver Stain for Mass Spectrometry performed better than or equal to the alternative silver staining method and GelCode Blue Stain.

2-D Gel Analysis of Rat Mitochondrial Preparation. A preparation of rat mitochondria were extracted, loaded and separated under identical 2-D gel conditions. All gels were run in a pH 5-8 gradient. Each gel was stained with SilverSNAP Stain for Mass Spectrometry (shown), Competitor I and GelCode Blue Stain Reagent respectively. Ten spots were identified that stained well under all staining conditions. These spots were picked for in-gel digestion and MS analysis.