Specialized Protein Gels Support — Troubleshooting
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One potential explanation is that the protein sample is getting re-oxidized before the run is complete. Reduced samples tend to oxidize more in the Tricine system. Adding more reducing agent will not solve the problem. One option is to alkylate the sample by reducing with 20 mM DTT at 70 degrees C for 30 minutes, followed by 50 mM iodoacetic acid. Another method which inhibits oxidation is the addition of thioglycolic acid to the running buffer. The reference to this is described by Hunkapiller et al., Methods in Enzymology, (91), 399, 1983. Caution should be taken when using this method since this compound is both toxic and expensive. In addition, the TGA must be fresh as it tends to get self-oxidized over time and will promote sample re oxidation.
If the Tricine gel is run with Tris-Glycine sample buffer, the bands will behave abnormally and resolve poorly. If the Tricine gel is accidentally run with Tris-Glycine running buffer, the gel will take longer to run and the resolution, especially for smaller proteins, will be worse than when the proteins are run on a Tris- Glycine gel with Tris-Glycine buffers. This is due to a combination of increase in stack area size (glycine is a slower ion than tricine) and the higher ionic strength of the Tricine gel.
In general, background staining in Tricine gels is slightly higher than in Tris-Glycine gels. The relatively higher concentration of solutes in Tricine gels as compared to their Tris-Glycine counter parts appears to slow down the rate of solution exchange into the gel. This can be counteracted by increasing the soak time in the second sensitization step (you may leave it in overnight) as per the modified procedure, and then proceed.
Some small proteases work better if the sample is heated for a short time before loading so the protease is denatured better and it doesn't digest the casein on its way through the gel. This may also help to improve the band sharpness.
During NativePAGE™ electrophoresis, it is common for the current to drop below 1 mA. Most power supplies register this as a “No Load” error and automatically shut off, resulting in the stopping of the gel run. This can be bypassed in some power supplies by disabling or turning off the “Load Check” feature.
V-shaped protein bands are caused by the presence of DNA in the sample. The artifact might be eliminated or minimized by shearing the DNA with additional sonication after the SDS-solubilization step. Alternatively, the DNA can be removed from the sample using an ultra-centrifuge.
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