VISUALIZATION OF MACROMOLECULES WITHIN GELS
Macromolecules within gels can be detected by a wide variety of techniques. Proteins can be stained with coomassie blue by soaking a gel in an acidic ethanolic solution that contains the dye. A more sensitive method for staining proteins is with silver nitrate. Below are protocols for perfoming Coomassie dye staining and silver staining.
If the macromolecules are radioactive, the gel will expose film or an image plate. Radioactive P32 can be incorporated into DNA by end-labelling with "hot" ATP (gamma-P32-ATP) and PNK (phosphonucleotide kinase) and into protein by hot ATP and serine/threonine or tyrosine kinases. DNA can can be stained by soaking with a solution of ethidium bromide and visualized using a UV light box. In general after staining, one must destain a gel to remove non-associated dye, which gives a high background.
Coomassie Staining Protocol
Coomassie staining is one of the simplest non-radioactive methods for visualizing proteins in gels.
Gel Silver Staining Protocol
Silver staining is the most sensitive method for detecting protein on a gel. The protocol as given here can be used for staining one or two gels.
Solution A: 40% ethanol
Solution B: Na2S2O3 (0.2g/L)
Solution C: 50mL water + 0.05g AgNO3
Solution D: Na2CO3 (30g/L)
Wash PAGE gel for:
1. 10 minutes in 50ml of Solution A + 25ml formaldehyde
2. 2 washes with water, 5 minutes each
3. 1 minute in 50 ml of Solution B
4. 2 washes with water, 20 seconds each
5. 10 minutes in Solution C
6. 50ml of Solution D (until the gel is developed, bands are visible)
Add 2.5ml of 50% citric acid to quench the reaction.