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November 01, 2010 (Vol. 30, No. 19)

Reproducible Laemmli PAGE Performance

Long Shelf Life Gels Designed to Improve Consistency, Speed, and Reproducibility

  • Conclusion

    TGX gels are currently available in 7.5%, 10%, 12%, 4–15%, 4–20%, and an Any kD gel formulation. Any kD gels offer resolution of proteins in the 10–250 kD range and can be used in screening experiments. These gels deliver superior resolution in the 20–100 kD range, the range most evaluated in 2-D samples, making it ideal for 2-D applications. Furthermore, all precast gels offered by Bio-Rad are manufactured without SDS and therefore, can be used for native PAGE applications.

  • Top Ten Tips to Improve Protein Gel Electrophoresis

    Over the last 15 years, Aldrin V. Gomes, Ph.D., assistant professor at the University of California, Davis, and his colleagues have routinely used gel electrophoresis in many of their experiments. Here are their top 10 tips that will help ensure great protein gel electrophoresis results:

    1 For the best resolution, protein samples should be vortexed before and after the heating step.

    2 Samples should not be boiled at 100ºC. Samples boiled at high temperatures can lead to high molecular weight aggregates. Samples should be heated at about 70ºC but less than 95ºC for 3–5 minutes for an SDS sample buffer preparation.

    3 Add 10 µL of 1x sample buffer to unused well lanes to avoid gel edge effects.

    4 SDS-PAGE running buffers can be reused two times without higher background signals. Reusing the buffer more than two times is possible, but running time increases.

    5 Sample loading buffer is important for the final resolution of samples. The most common mistake is not adding enough bromophenol blue making it difficult to see the sample when it is being loaded into the wells. To improve reproducibility and reliability, make 100 mL of Laemmli Sample Buffer and aliquot this into 1 mL fractions. Once validated, the batch of buffer solution can be used for over 500 gels. Sample buffer aliquots are stable for 6 months at -20ºC and >1 year at -80ºC.

    6 Only constant voltage gives constant protein mobility during gel electrophoresis, NOT constant power conditions.

    7 Double-check wells for damage and note the maximum volume that the well can hold. Overfilling wells can lead to artifacts.

    8 Remove aggregates and improve resolution by centrifuging all samples in a microfuge at >10,000 xg for two minutes prior to loading.

    9 Samples boiled in sample buffer can be aliquoted and stored at -20ºC for >4 weeks or at 4ºC for at least a week. Samples should be warmed at 37ºC for 1–2 minutes before
    use. Repeated freeze-thawing can lead to protein degradation.

    10 High salt concentrations cause gel artifacts. If the salt concentration of a sample is high, the protein can be concentrated with 10% (w/v) trichloroacetic acid (TCA) (incubate for five min. at 4ºC). The precipitated protein is collected by centrifugation and the pellet washed with cold acetone. The pellet is then resuspended in an appropriate buffer. If the sample concentration is too dilute, then TCA could also be used to concentrate these samples.

    For more detailed tips, visit www.lablife.org/lab?groupid=2441 for a Protein Gel Electrophoresis Tips and Troubleshooting Guide.

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