There are two primary types of Western blot gels you can use during your experiments: native and SDS-PAGE gels. Keep in mind that one is slightly different from the other. Because these variations are vital to the success of your research, it is important to understand which gel will best fit your target proteins and molecular weight range.
What Is a Native Gel?
A native gel separates proteins based on their size and charge. The proteins remain in their native state and are not denatured during the process. Since proteins are not reduced or denatured in this assay, native gels are typically used to investigate protein complexes or enzymatic activity.What Is an SDS-PAGE Gel?
An SDS-PAGE gel separates proteins based on their size. The proteins are denatured during the process by a detergent called sodium dodecyl sulfate (SDS). This type of assay is more commonly selected than native gels due to its simplicity. SDS-PAGE gels can be used when the estimated molecular weight of a denatured protein together with primary antibody affinity is sufficient for protein detection.Types of SDS-PAGE Gels
Additionally, there are two different types of SDS-PAGE gels: gradient and fixed gels. These differ on acrylamide concentration and pore size and are specific to certain molecular weight ranges.
Gradient Gels
Fixed Gels
A gradient gel is comprised of a single layer with an acrylamide gradient that increases in concentration from top to bottom. Therefore, its pores are larger at the top with a gradual reduction in size toward the bottom. It is efficient at separating proteins with a broad range of molecular weights.
A fixed gel is comprised of two distinct layers: a stacking (top) gel layer with a lower acrylamide concentration and a resolving (bottom) gel layer with a higher acrylamide concentration. Its pores are uniform in size for each layer, and it is efficient at separating proteins with similar molecular weights.
Depending on the needs of your research, a combination of gradient and fixed gels can also be used. To do so, separate your samples with a complex mixture of proteins on a gradient gel first to narrow down the molecular weight range. Next, extract them from the gradient gel and separate them again on a fixed gel to develop better resolution between similar molecular weight proteins.
Western Blot Gels
Know the differences but not sure where to find the gels you need? Fixed and gradient gels can either be poured in the lab or purchased pre-cast. If you pour the gels yourself, it is easier to change acrylamide gel percentage. Pre-cast gels are available in a wide variety of acrylamide gel percentages, but each type must be purchased.
Take your experiment one step further and watch the Preparing the Gel learning path on the Lambda U® Education Portal to optimize your gel preparation and practices. If you ever have questions about types of Western blot gels and when to use them, please feel free to contact us at your convenience. We'd love to walk you through your options and help you find the best solution for your protocol.
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