Lrg:Silver staining

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Contents

Overview

Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) whilst using very simple and cheap equipment and chemicals.

Materials

  • Glacial acetic acid
  • Methanol
  • distilled ethanol
  • Formaldehyde
  • Na2S2O3 ( sodium thiosulphate)
  • AgNO3 (Silver nitrate)
  • Na2CO3 ( sodium carbonate)
  • tripple distilled water or milliQ water

( this protocol is optimised for 10×10 size gels


Procedure

  1. Fixing. (40-60 Min)
    1. 6 mL of acetic acid
    2. 25 mL of methanol
    3. 0.25 mL of HCHO

makeup the volume to 100ml. and fix the gel in this solution for the indicated time( fixation gets rid of interfering compounds and fixes the protein by precipitation)

  1. Dehydration. 30 min(2 times)
    1. 50 % ethanol
  2. pretreatment or sensitization and rinses to increase the sensitivity and contrast of the staining
    1. 20 mg Na2S2O3 ( sodium Thiosulphate) in 100 mL tripple distilled water for 1 min
  3. wash with tripple distilled water 3 times
  4. Impregnation (20 min)
    1. 200 mg of AgNO3 and 25 μL of HCHO in 100 mL of tripple distilled water for 20 min
  5. Developing
    1. 6 g of Na2CO3 + 75 μL of HCHO + 1 mg of Na2S2O3
    2. incubate till the bad appears
  6. wash the gel three times with distilled water
  7. storage and stop solution
    1. 6 ml of acetic acid + 2.5 mL of methanol makeup the volume to 100mL with distilled water

Notes

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References

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