Biological Mass Spectrometry Protocols

Silver staining of polyacrylamide gelד


  • Fixation solution (50 : 5 : 45 v/v/v methanol : acetic acid : water)
  • Sensitizing solution (0.02 % sodium thiosulfate)
  • 0.1 % AgNO3
  • Developing solution (0.04 % formaldehyde in 2 % sodium carbonate)
  • 1 % acetic acid


  1. After the gel has been run, fix the protein by incubating the gel slab in fixation solution for 20 - 30 minutes.
  2. Rinse the gel slab with water (2 changes, two minutes per change) and then leave it further in water for one hour on a shaking platform.
  3. Sensitize the gel with sensitizing solution for 1 - 2 minutes. Discard solution and quickly rinse the gel slab with two changes of water (10 seconds each).
  4. Incubate the gel in chilled 0.1 % AgNO3 for 30 minutes at 4oC (fridge).
  5. Discard silver nitrate solution and quickly rinse the gel with two changes of water (30 seconds per each change).
  6. Develop the gel with developing solution. Discard the developing solution as soon as it turns yellow and replace it with a fresh portion.
  7. When a sufficient degree of staining has been obtained, quench staining by discarding the developing solution and replacement with 1 % acetic acid. Wash the gel with 1 % acetic acid several times and store in the same solution.
  8. Extended washing time helps to eliminate yellowish background usually observed after long developing of the gel (see step 6 of this protocol)
  9. Agitate gently to make sure that the gel slab is covered evenly.
  10. Silver stained gels can be stored at 4o C in 1 % acetic acid for months. In some cases the color of the stained protein bands might slightly change in time. However these changes do not affect the results of mass spectrometric sequencing.

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Mass Spectrometry Sample preparation for ESI-MS

Before electrospray ionization (ESI) mass spectrometry (MS) sample was passed over a micro-column consisting of about 300nl Poros R1 reversed phase material (ABI). Purified protein was eluted directly into a nano-electrospray capillary (Proxion, Denmark) using 5ul of 70% acetonitrile/5% formic acid.

  1. Mass Spectrometry analysis was performed in Biological Mass Spectrometry Unit at Weizmann Institute of Science, Department of Biological Services
  2. In-gel Digestion and Protein Identification by LC-ESI-MS/MS

    In-gel Digestion Protein bands were excised from the SDS gel stained with Gel Code and distained using multiple washings with 50% acetonitrile in 50 mM ammonium bicarbonate. The protein bands were subsequently reduced, alkylated and in-gel digested with bovine trypsin (sequencing grade, Roche Diagnostics, Germany), at a concentration of 12.5 ng/ul in 50 mM ammonium bicarbonate at 370C, as described (1). The peptide mixtures were extracted with 80% CH3CN, 1% CF3COOH, and the organic solvent was evaporated in a vacuum centrifuge. The resulting peptide mixtures were reconstituted in 80% Formic Acid and immediately diluted 1:10 with Milli-Q water prior to the analysis by online reversed-phase nano-LC (liquid chromatography) - electrospray ionization (ESI) tandem mass spectrometric analyses (MS/MS).
    Nano-LC-ESI-MS/MS Samples were analyzed in LTQ-Orbitrap (Thermo Fisher Scientific, Bremen, Germany) operated in the positive ion mode. Nano-LC-ESI-MS/MS - Peptide mixtures were separated by online reversed-phase nanoscale capillary LC and analyzed by ESI-MS/MS. For the LC-MS/MS, the samples were injected onto an in-house made 15 cm reverse d phase spraying fused-silica capillary column (inner diameter 75 m, packed with 3 m ReproSil-Pur C18 A18 media (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany), using an UltiMate 3000 Capillary/Nano LC System, consisting of FamosTMMicro Autosampler, SwitchosTM Micro Column Switching Module (LC Packings, Dionex). The LC setup was connected to the LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a nanoelectrospray ion source (Thermo Fisher Scientific, Bremen, Germany). The flow rate through the column was 250 nL/min. An acetonitrile gradient was employed with a mobile phase containing 0.1% and 0.2% formic acid in Milli-Q water in buffers A and B, respectively. The injection volume was 5 ul. The peptides were separated with 50 min gradients from 5 to 65% CH3CN. In the nano-electrospray ionization source, the end of the capillary from the nano-LC column was connected to the emitter with pico-tip silica tubing, i.d. 20 um (New Objective) by stainless steel union, with a PEEK sleeve for coupling the nanospray with the on-line nano-LC. The voltage applied to the union in order to produce an electrospray was 2.4 kV. Helium was introduced as a collision gas at a pressure of 3 psi. The mass spectrometer was operated in the data-dependent mode. Survey MS scans were acquired in the Orbitrap with the resolution set to a value of 60,000. Up to the 7 most intense ions per scan were fragmented and analyzed in the linear trap. For the analysis of tryptic peptides, survey scans were recorded in the FT-mode followed by data-dependent collision-induced dissociation (CID) of the 7 most intense ions in the linear ion trap (LTQ). Raw spectra were processed using open-source software DTA SuperCharge (http :// The data were searched with MASCOT (Matrix Science, London, UK) against a Swissprot or NCBI database. Search parameters included variable modifications of 57.02146 Da (carboxyamidomethylation) on Cys, 15.99491 Da (oxidation) in Met and 0.984016 Da (deamidation) on Asn and Gln. The search parameters were as follows: maximum 2 missed cleavages, initial precursor ion mass tolerance 6 ppm, fragment ion mass tolerance 0.6 Da. The identity of the peptides were concluded from the detected collision-induced dissociation products by Mascot, Protein Discoverer (Thermo Fisher Scientific LTD), confirmed by Scaffold Software (Proteome Software, Inc) and confirmed by manual inspection of the fragmentation series. References: 1. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1966) Anal. Chem. 68, 850- 858.

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