Novex™ 10 bis 20 %, Tricin, 1,0 mm, Mini Protein Gele

There may be too much beta-mercaptoethanol (BME), sample buffer salts, or dithiothreitol (DTT) in your samples. If the proteins are over-reduced, they can be negatively charged and actually repel each other across the lanes causing the bands to get narrower as they progress down the gel.

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A temperature increase to 35°C to 40°C during electrophoresis is not uncommon for Tricine gels. If you want to run the gels at a cooler temperature, the lower (outer) buffer chamber can be filled higher or they can be run at a lower voltage, for example 100 V.

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For non-sequencing applications, any transfer buffer used with Tris-Glycine gels can be used with Tricine gels including Tris-Glycine transfer buffer. For sequencing applications, the buffer should be chemically compatible with sequencing protocols. Non-glycine based transfer buffers such as the NuPAGE Transfer buffer, 1/2X TBE Transfer buffer, or CAPS Buffer can be used for N-terminal sequencing . Generally, a pH which is close to neutral is desirable to maintain gel and protein stability. High current should be avoided because it can lead to heat generation and instability.

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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.

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Protein samples are possibly reoxidizing before the run is complete in the Tricine gel system. Since Tricine is a glycine derivative, the running pH ranges of the two systems are different. As a consequence, 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°C for 30 min, followed by 50 mM iodoacetic acid to alkylate.

Another method which inhibits oxidation is the addition of thioglycolic acid (TGA) to the running buffer. The reference to this is described by Hunkapiller et al, Methods of 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 become oxidized itself over time. Oxidized TGA will actually promote sample re-oxidation.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.