Silver Staining

1. Silver Staining

2. Why silver stain? • Silver stain has high sensitivity compared to other stains. • Silver stain reveals • subnanogram quantities of ss and ds DNA • 2-5 ng protein • Ethidium bromide reveals • 10 ng ds DNA • Coomassie blue reveals • >= 60 ng protein • Equally or more sensitive methods • take more than one hour or • are more expensive.

3. How does silver staining work? • Reduction of Ag+ to metallic Ag  stain • Ag+ ions complex with • bases of DNA. • sulfhydryl and carboxyl groups of proteins. • Ag+ complexed with DNA or protein is selectively reduced. • Ag+ in solution is more slowly reduced than is complexed Ag+. • A process to increase selectivity for complexed Ag+ is incorporated into Bio-Rad’s Silver Stain Plus kit. • Gottlieb and Chavko (1986) Analytical Biochemistry 165, 33-37.

4. Components of the Bio-Rad Silver Stain Plus (SS+) Process • Fixative enhancer solution • H2O purified by filtration through ion-exchange resins and organic trapping resins • Staining solution • Stop solution There is more about each of these on the following slides.

5. Components of the SS+ ProcessFixative enhancer solution • Methanol • Acetic acid • acetic acid/methanol • fixes DNA or protein in place • prevents gel itself from staining too darkly • Fixative enhancer concentrate = glycerol • somehow enhances fixation; may minimize shrinkage of the gel by methanol

6. Components of the SS+ ProcessH2O • H2O purified by filtration through ion-exchange and organic trapping resins • Removes solutes from gel to prevent high background • Offending solutes include • impurities in acrylamide • glycerol • urea • glycine • Triton X-100 (SS+ works in the presence of Triton X-100) • agarose

7. Components of the SS+ ProcessStaining solution • Employs a carrier-complex chemistry • Permits delivery of silver ions to DNA or protein bands in the gel in a reducing environment without • precipitation in solution or • reduction in the gel outside of the bands • Includes • silver complex solution (AgNO3 and NH4NO3) • reduction moderator solution (tungstosilicic acid (TSA)) • image development reagent (formaldehyde) • development accelerator reagent (Na2CO3)

8. Components of the SS+ ProcessStaining solution (cont’d) • Silver complex solution (AgNO3 and NH4NO3) • provides the Ag+ that is reduced to Ag • just over 2 NH4NO3 to 1 AgNO3 • Reduction moderator solution • H2O solution of dodecatungstosilicic acid (TSA), a heteropoly acid (H4O40SiW12) • Similar to an ion exchange bead • Serves as carrier for the Ag+ complex so that it is not free in solution. • This is important!!

9. Components of the SS+ ProcessStaining Solution (cont’d) • Image developer reagent (formaldehyde) • Formaldehyde is a reducing agent in alkaline conditions. • It is oxidized to formic acid. • It will not reduce Ag+ bound to tungstosilicic acid as rapidly as it will reduce unbound ions.

10. Components of the SS+ ProcessStaining Solution (cont’d) • Developer accelerator reagent (Na2CO3) • Promotes all the chemistry in the stain • Provides alkaline conditions • Deprotonates the TSA to form Ag+ binding sites in the acid • Deprotonates the NH4+ NH3 • NH3 complexes with Ag+ • In complex, Ag+ can’t be precipitated by the CO32- • Ag2CO32- is a white precipitate • Complex binds to tungstosilicic acid • Activates the formaldehyde  powerful reducing agent for Ag+ not bound to TSA

11. After the mixed staining solution is applied to the gel . . . . . • At first, almost no metallic silver is formed • at the locations of DNA or protein • in solution • in the gel matrix • But, nucleophilic, aromatic, and heteroaromatic groups in proteins and DNA • readily compete with TSA to form complexes with Ag+ and • Ag+ in complex with DNA or protein is not protected from reduction by formaldehyde

12. So . . . • Silver complexed with DNA or protein is rapidly reduced • An autocatalysis occurs • Ag facilitates reduction of nearby Ag+ • And after a while . . . . . ., as if out of nowhere . . . . ., bands appear with almost no background ! ! !

13. Imagine . . . . • The incredible balance required between • The acidity of the TSA and its buffering capacity vs. • The need to have carbonate create basic conditions to • activate the formaldehyde • deprotonate the NH4+

14. Components of the Silver Stain Process • Stop solution (5% acetic acid) • Stops the reduction

15. Safety Precautions • Wear gloves, glasses, lab coat. • Handle Image Development Reagent carefully. • Use in areas of good ventilation. • Avoid breathing vapors. • Avoid contact with skin. • In case of contact with eyes, flush with copious amounts of water and contact a physician.

16. Success Precautions • Prevent Ag2CO3 precipitate by adding Na2CO3 quickly and all at once. • Adding it slowly prevents formation of NH3 and the NH3/Ag+ complex and allows free Ag+ to precipitate with the excess of CO3= • If too much precipitate is allowed to form, it may not go back into solution. • The Ag+ in the precipitate is readily reduced  background from autocatalysis. The precipitate is sticky.

17. Success Precautions • Be sure the gel is submerged and moving freely during exposure to all solutions. • Avoid orbital, as opposed to oscillating, shakers. Orbital shakers  vortex, which causes concentration gradients to develop from the central portion of the gel to the outside. • Avoid microheterogeneities in the surface of the dish or gel. They trigger the reduction chemistry. • cracks and compressions in the gel • scratches in the container

18. Success Precautions (cont’d) • Use freshly made CO3= solutions. • carbonate solutions tend to dissolve CO2 from the air  HCO3- which causes any precipitates which form to be more problematic. • Avoid conjugated unsaturations; they complex with Ag+ • found in polyester, polycarbonate, plasticized (flexible) polypropylene or polyethylene • Plasticizers are often esters of phthallic acid, which is aromatic, and so has conjugated unsaturations.

19. Thanks to Bio-Rad tech support for providing details of the reaction not included with the kit!!!!