Silver Staining SDS-PAGE Gels

Method 1

  1. Make 7% Acetic Acid by mixing 186 ml of distilled H2O with 14 ml of glacial acetic acid.

  2. Make 50% Methanol by mixing 200 ml of distilled H2O with 200 ml of methanol. Optional - for extra fixation/crosslinking add 240 ul of 50% glutaraldehyde to the 50% methanol (makes solution 0.03% glutaraldehyde).

  3. Soak gel in 7% acetic acid for 7 minutes.

  4. Soak gel in 200 ml of 50% methanol for 20 minutes.

  5. Soak gel in 200 ml of 50% methanol for 20 minutes.

  6. Prepare Solution A by adding 0.8 g of silver nitrate to 4 ml of distilled H2O.

  7. Rinse gel in 200 ml distilled H2O for 10 minutes.

  8. Rinse gel in 200 ml distilled H2O for 10 minutes.

    Note: Steps 7 and 8 are very important for the NuPAGE gels if you skip these steps or do not rinse the gel for long enough the gel will develop too quickly and have significantly more background.

  9. 5 minutes before end of final distilled H2O rinse prepare solution B by adding 21 ml of distilled H2O with 250 ul of 30% NaOH (to make 0.36%) + 1.4 ml of 14.8M ammonium hydroxide.

  10. Make staining solution by adding solution A to solution B dropwise while stirring, then add 76 ml of distilled H2O.

  11. Soak gel in the staining solution for 15 minutes.

  12. Rinse gel in 200 ml distilled H2O for 5 minutes.

  13. Rinse gel in 200 ml distilled H2O for 5 minutes.

  14. Make developing solution by mixing 200 ml of distilled H2O with 1 ml of 1% citric acid and 100 ul of 37% formaldehyde.

  15. Soak gel in developing solution until bands are visible, usually 2 to 15 minutes.

  16. Stop development by rinsing gel with 3 changes of 200 ml distilled H2O.

    The sensitivity of this method should be in the 10ng/band range.

Method 2 (Rapid)

  1. Make fixing solution by adding 50 ml methanol and 10 ml glacial acetic acid to 40 ml distilled H20 (50% methanol, 10% acetic acid).

  2. Add gel to fixing solution and microwave on high for 1 minute, then shake 15 minutes.

  3. Wash gel with 100 ml distilled H20.

  4. Microwave gel in 100 ml distilled H20 on high for 1 minute, then shake 10 minutes (or until the gel is rehydrated).

  5. Add gel to 100 ml reducing solution, which contains 5 ug/ml DTT in distilled H20.

  6. Microwave on gel in 100 ml reducing solution on high for 1 minute, then shake 15 minutes (or until the gel has cooled).

  7. Stainthe gel with 100 ml 0.1% AgNO3 in distilled H20.

  8. DO NOT MICROWAVE. Shake 15 minutes.

  9. Wash gel twice with distilled H20. Shake while washing.

  10. Make frsh developer solution by adding 40 ml 15% Na2CO3 to 160 ml distilled H20).

  11. Wash gel twice with 50 ml developer solution.

  12. Add 100 ul of 37% formaldehyde to the remaining 100 ml of developer solution.

  13. Pour developer solution on gel and shake until bands are seen.

  14. STOP by adding 5 ml 2.3 M citric acid (should be bubbling).

  15. Shake in STOP for 10 minutes, then wash out with distilled H20 several times (otherwise the background turns yellow).

  16. Soak in 5% glycerol for 15 minutes or longer before drying gel.

    If preparing a sample for mass spectrometry, you may wish to use the protocol found here.