Presentation on theme: "Silver Staining. Why silver stain? n Silver stain has high sensitivity compared to other stains. u Silver stain reveals F subnanogram quantities of ss."— Presentation transcript:
Why silver stain? n Silver stain has high sensitivity compared to other stains. u Silver stain reveals F subnanogram quantities of ss and ds DNA F 2-5 ng protein u Ethidium bromide reveals F 10 ng ds DNA u Coomassie blue reveals F >= 60 ng protein n Equally or more sensitive methods u take more than one hour or u are more expensive.
How does silver staining work? n Reduction of Ag + to metallic Ag stain n Ag + ions complex with u bases of DNA. u sulfhydryl and carboxyl groups of proteins. n Ag + complexed with DNA or protein is selectively reduced. u Ag + in solution is more slowly reduced than is complexed Ag +. u A process to increase selectivity for complexed Ag + is incorporated into Bio-Rad’s Silver Stain Plus kit. F Gottlieb and Chavko (1986) Analytical Biochemistry 165, 33-37.
Components of the Bio-Rad Silver Stain Plus (SS+) Process n Fixative enhancer solution n H 2 O purified by filtration through ion-exchange resins and organic trapping resins n Staining solution n Stop solution There is more about each of these on the following slides.
Components of the SS+ Process Fixative enhancer solution n Methanol n Acetic acid u acetic acid/methanol F fixes DNA or protein in place F prevents gel itself from staining too darkly n Fixative enhancer concentrate = glycerol u somehow enhances fixation; may minimize shrinkage of the gel by methanol
Components of the SS+ Process H 2 O n H 2 O purified by filtration through ion-exchange and organic trapping resins u Removes solutes from gel to prevent high background u Offending solutes include F impurities in acrylamide F glycerol F urea F glycine F Triton X-100 (SS+ works in the presence of Triton X-100) F agarose
Components of the SS+ Process Staining solution n Employs a carrier-complex chemistry u Permits delivery of silver ions to DNA or protein bands in the gel in a reducing environment without F precipitation in solution or F reduction in the gel outside of the bands n Includes u silver complex solution (AgNO 3 and NH 4 NO 3 ) u reduction moderator solution (tungstosilicic acid (TSA)) u image development reagent (formaldehyde) u development accelerator reagent (Na 2 CO 3 )
Components of the SS+ Process Staining solution (cont’d) n Silver complex solution (AgNO 3 and NH 4 NO 3 ) u provides the Ag+ that is reduced to Ag u just over 2 NH 4 NO 3 to 1 AgNO 3 n Reduction moderator solution u H 2 O solution of dodecatungstosilicic acid (TSA), a heteropoly acid (H 4 O 40 SiW 12 ) F Similar to an ion exchange bead F Serves as carrier for the Ag+ complex so that it is not free in solution. This is important!!
Components of the SS+ Process Staining Solution (cont’d) n Image developer reagent (formaldehyde) u Formaldehyde is a reducing agent in alkaline conditions. u It is oxidized to formic acid. u It will not reduce Ag + bound to tungstosilicic acid as rapidly as it will reduce unbound ions.
Components of the SS+ Process Staining Solution (cont’d) n Developer accelerator reagent (Na 2 CO 3 ) u Promotes all the chemistry in the stain u Provides alkaline conditions F Deprotonates the TSA to form Ag + binding sites in the acid F Deprotonates the NH 4 + NH 3 NH 3 complexes with Ag + In complex, Ag+ can’t be precipitated by the CO 3 2- –Ag 2 CO 3 2- is a white precipitate Complex binds to tungstosilicic acid F Activates the formaldehyde powerful reducing agent for Ag+ not bound to TSA
After the mixed staining solution is applied to the gel..... n At first, almost no metallic silver is formed u at the locations of DNA or protein u in solution u in the gel matrix n But, nucleophilic, aromatic, and heteroaromatic groups in proteins and DNA u readily compete with TSA to form complexes with Ag + and u Ag + in complex with DNA or protein is not protected from reduction by formaldehyde
So... n Silver complexed with DNA or protein is rapidly reduced n An autocatalysis occurs u Ag facilitates reduction of nearby Ag+ n And after a while......, as if out of nowhere....., bands appear with almost no background ! ! !
Imagine.... n The incredible balance required between u The acidity of the TSA and its buffering capacity vs. u The need to have carbonate create basic conditions to F activate the formaldehyde F deprotonate the NH4 +
Components of the Silver Stain Process n Stop solution (5% acetic acid) u Stops the reduction
Safety Precautions n Wear gloves, glasses, lab coat. n Handle Image Development Reagent carefully. u Use in areas of good ventilation. u Avoid breathing vapors. u Avoid contact with skin. u In case of contact with eyes, flush with copious amounts of water and contact a physician.
Success Precautions n Prevent Ag 2 CO 3 precipitate by adding Na 2 CO 3 quickly and all at once. u Adding it slowly prevents formation of NH 3 and the NH 3 /Ag+ complex and allows free Ag+ to precipitate with the excess of CO 3 = u If too much precipitate is allowed to form, it may not go back into solution. u The Ag + in the precipitate is readily reduced background from autocatalysis. The precipitate is sticky.
Success Precautions n Be sure the gel is submerged and moving freely during exposure to all solutions. n 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. n Avoid microheterogeneities in the surface of the dish or gel. They trigger the reduction chemistry. u cracks and compressions in the gel u scratches in the container
Success Precautions (cont’d) n Use freshly made CO 3 = solutions. u carbonate solutions tend to dissolve CO 2 from the air HCO 3 - which causes any precipitates which form to be more problematic. n Avoid conjugated unsaturations; they complex with Ag+ u found in polyester, polycarbonate, plasticized (flexible) polypropylene or polyethylene F Plasticizers are often esters of phthallic acid, which is aromatic, and so has conjugated unsaturations.
Thanks to Bio-Rad tech support for providing details of the reaction not included with the kit!!!!