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Protein detection

The application of the 2-D PAGE technology to separate, analyse and characterize proteins contained in biological samples would not have been possible without the development of complementary detection methods. Perhaps today one of the most popular non-radioactive protein detection is the silver staining which is 100-fold more sensitive than Coomassie Brilliant Blue staining [6, 14, 16-19]. The different masters shown in SWISS-2DPAGE were stained with the ammoniacal silver staining as followed:

Silver staining protocol

All steps were performed on an orbital shaker at 36 rpm [6].

  1. At the end of the second dimension run, the gels were removed from the glass plates and washed in deionized water for 5 min.
  2. Soaked in ethanol: acetic acid: water (40: 10: 50) for 1 hour.
  3. Soaked in ethanol: acetic acid: water (5: 5: 90) for 2 hours or overnight.
  4. Washed in deionized water for 5 min.
  5. Soaked in a solution containing glutaraldehyde (1%) and sodium acetate (0.5 M) for 30 min.
  6. Washed 3 times in deionized water for 10 min.
  7. In order to obtain homogeneous dark brown staining of proteins, gels were soaked twice in a 2,7 naphtalene-disulfonic acid solution (0.05% w/v) for 30 min.
  8. Rinsed 4 times in deionized water for 15 min.
  9. Gels were stained in a freshly made ammoniacal silver nitrate solution for 30 minutes. To prepare 750 ml of this solution, 6 g of silver nitrate were dissolved in 30 ml of deionized water, which was slowly mixed into a solution containing 160 ml of water, 10 ml of concentrated ammonia (25%) and 1.5 ml of sodium hydroxide (10 N). A transient brown precipitate might form. After it cleared, water was added to give the final volume.
  10. After staining, the gels were washed 4 times in deionized water for 4 min.
  11. The images were developed in a solution containing citric acid (0.01% w/v) and formaldehyde (0.1% v/v) for 5 to 10 min.
  12. When a slight background stain appeared, development was stopped with a solution containing Tris (5% w/v) and acetic acid (2% v/v).

Scanning

The Laser Densitometer (4000 x 5000 pixels; 12 bits/pixel) from Molecular Dynamics and the GS-700 from Bio-Rad have been used as scanning devices. This scanners were linked to Sparc workstations and Macintosh computers.



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