Fluorescent Silver Staining: A Technique to Visualize Total Protein in Polyacrylamide Gels
Transcript
Fluorescent silver staining of proteins in polyacrylamide gels employs fluorogenic probes to detect and quantify the proteins.
Begin with a polyacrylamide gel with proteins, separated into bands through electrophoresis. Soak in a fixative mixture of ethanol and acetic acid, which prevents the bands from diffusing by denaturing the proteins.
Wash the gel to remove residual acetic acid and ethanol. Immerse the gel in silver nitrate stain. Silver ions bind strongly to negatively charged groups in proteins.
Incubate in the dark. Rinse the gel in ultrapure water to remove unbound silver ion complexes.
Immerse the gel in a developing solution containing a multi-component fluorogenic probe – TPE-4TA. This anionic probe targets silver ions bound to the protein, forming insoluble aggregates that activate the fluorogenic properties of the probe, thus imparting fluorescence.
As the fluorescence activation is aggregation dependent, the unbound probes do not emit any signal, enabling total protein staining with reduced background emission.
Incubate the gel in the dark with gentle agitation, ensuring complete fluorogenic development. Transfer the gel in a destain buffer to remove unbound probes. Finally, image the gel to obtain the band signal intensity. Plot the fluorescence signal intensity against standard protein curve to calculate the total amount of proteins in the sample.
