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Most people know that gel electrophoresis separates proteins based on charge and size. But the type of gel you run really determines how your proteins are separated and can affect your outcome. Denaturing and native gels are not interchangeable.

Here’s your quick guide to choosing the one that’s right for your experiment.

Denaturing it

Sample loading buffer for denaturing gels has two key components:

  • The strong detergent, sodium dodecyl sulfate (SDS) and
  • A reducing agent, either dithiothreitol (DTT) or beta-mercaptoethanol (β-Me).

After these are mixed with your sample, you heat it up.

The two chemicals plus heat work together to make the charge of the proteins negligible allowing them to be separated mainly by size.

How it works

The reducing agent breaks disulfide bonds disturbing the higher complex structure of the protein, while SDS coating of proteins confers a net negative charge to the proteins.  Add some heat and denaturation becomes more efficient as SDS can access all areas of the protein more easily, including hydrophobic pockets.

The end result is a mixture containing linear proteins that will migrate through the gel based on their molecular weight – higher molecular weight proteins will move more slowly while lower molecular weight proteins will move quickly.

Therefore, use denaturing gels for:

  • Estimating/confirming the molecular weight of a protein
  • Isolation of proteins based on size
  • Separation of protein complexes into individual components
  • Estimating purity level of a sample
  • Western blotting
  • Preparation for protein sequencing

Going native

If you are going native, then things are a bit easier for you.  Native gels do not use SDS or a reducing agent in the sample loading buffer.   And SDS is not used in the electrophoresis buffer.  You also don’t heat the samples prior to loading them on the gel.  You’re not being a slacker – going native helps maintain the protein’s secondary structure and interactions within protein complexes often preserving enzymatic activity.

How it works

Most proteins have a net negative charge at slightly basic pH.  Proteins that are more negative migrate faster through the gel while those that are less negative will migrate more slowly.  In addition, the gel matrix retards protein movement based on the size of the protein (bigger proteins move more slowly).  Therefore native gels separate proteins based on both charge and mass.

Use native gels for:

  • Determining the aggregation state of a protein
  • Isolation of enzymes
  • Studying protein complexes

Whether you are going native or denaturing your samples, remember to prepare your samples carefully to avoid degradation.

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