Quick, Easy and Cheap Protein Visualization
by Ivan Spasojevic Labtimes 05/2015
Tryptophans are not the most abundant of protein residues. But you don’t need a lot of tryptophans for staining proteins in polyacrylamide gels.
Potassium ferricyanide (III) oxidises Trp at 25 °C and high pH.
Native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) is one of the most frequently applied techniques in life science labs. Following PAGE, protein bands are commonly visualized using the Coomassie Brilliant Blue (CBB) protocol, silver staining, or different fluorescent stains.
Each of these staining procedures, however, fails to meet at least one of the dream features from the title: CBB is not quick (although the overnight staining is rather popular), silver is everything but easy (e.g. very sensitive) and well-performing fluorescent stains are not cheap, at all. So, novel approaches for in-gel protein visualization are more than welcomed.
My group at the Department of Life Sciences, Institute for Multidisciplinary Research, University of Belgrade, looked for a new way to employ proteins’ constituent fluorophores in visualization and came up with a method that relies on the oxidation of tryptophan (Trp) residues (Pristov et al., Anal. Biochem. 480, 6-10). We tested several oxidising agents and tuned the method using different concentrations, incubation times, and pH values.
The best performance was obtained for the following protocol:
- Place gel in water solution of K3[Fe(CN)6] (100 mM) and NaOH (1 M).
- Incubate it in the dark at room temperature for 30 min.
- Transfer the gel to water (no washing is needed) and voilà, it is ready for scanning.
Potassium ferricyanide is light-sensitive, so the solution should be freshly prepared for staining and kept in the dark. It is also important to have in mind that a high concentration of NaOH provokes protein denaturation (in order to bring “buried” Trp residues to the surface). A short exposure of stained gels to light as well as repetitive scanning, do not have negative effects on sensitivity.
Fluorophore that is produced via ferricyanide-provoked Trp oxidation shows maximal excitation/emission at 345/460 nm. At the moment, this would require non-standard equipment.
Fortunately, the fluorophore can be excited with wavelengths between 300 to 450 nm, with emission in the 420 to 550 nm range; these settings are met by a number of commercial gel scanners. With excitation/emission at 395/525 nm and bovine serum albumin (3 Trp per chain) as a standard, the sensitivity was comparable to CBB and about 3.5 times lower compared to the optimal settings. In other words, under optimal scanning settings, the sensitivity of ferricyanide staining equals the sensitivity of CBB multiplied by the number of Trp per chain. For example, if you invest a lot of time isolating some protein that is luckily Trp-rich, the application of ferricyanide staining can save much of your precious sample.
Native PAGE gel of human plasma (5 µL per lane) stained with ferricyanide or CBB. Photo: Ivan Spasojevic
Band fluorescence intensity is directly proportional to the number of Trp in the band. And here lays yet another advantage with this method: it can be useful in Trp quantification. Calibration fit for band fluorescence intensity and the number of Trp (nmol) per band is created using equimolar (subunit) amounts of a set of proteins with different number of Trp per chain/subunit. Another calibration fit is prepared for band intensity and the amount of proteins (µg) using CBB staining.
With these two, the amount of Trp per µg of protein can be calculated for experimental gels. This might be useful in the process of identifying proteins in biological samples, by narrowing down the list of ‘‘suspects’’ according to the number of Tryptophans.
Of note: if, at some point, you are not satisfied with the results of this staining protocol, the gel can undergo post-staining with CBB without any loss of sensitivity.
Last Changed: 14.09.2015