SDS-PAGE

Gel Systems

Different Gel systems for SDS PAGE are available. In general, they differ in percentage and buffer composition directly influencing their resolving properties.

• Gel percentage refers to the total concentration of acrylamide and bis-acrylamide in the gel. It directly determines pore size and therefore the molecular weight range of proteins that can be resolved.
• Low-percentage gels (3–8%) are well suited for separating high–molecular-weight proteins (>100 kDa) but exhibit limited resolution for small proteins.
• Medium-percentage gels (10–12%) have a balanced pore size and are a good choice for most proteins (20–100 kDa).
• High-percentage gels (15–20%) have a small pore size and are optimal for low–molecular-weight proteins (<20–30 kDa). However, they show high resistance to migration of large proteins.
• Gradient gels contain a continuous increase in acrylamide concentration from top to bottom (e.g., 4–20% or 3-8%). They can separate wider molecular weight ranges in a single gel with high resolution for both small and large proteins.

Buffer Systems

Buffer systems control pH, ion composition, and conductivity, which strongly influence protein migration, resolution, and separation range.

• Tris–Glycine Gels are the most widely used SDS-PAGE system with a resolving gel pH: ~8.8. The pH shift between the stacking and the resolving gel is not stable over time making long-term storage impossible. They are well suited for the separation of mid-range proteins but are less optimal for very small and very large proteins.
• Tris–Tricine Gels are designed to improve separation of small proteins and peptides (<10–15 kDa). Tricine replaces glycine as the trailing ion and allows better stacking and resolution of low-MW proteins
• Bis–Tris Gels operate at near-neutral pH (~6.4). They produce sharper bands and proteins are less prone to modifications. Precast gels are often of this type since they are very stable and allow long-term storage. Bis-Tris gels are compatible with different running buffers (e.g., MOPS or MES) to tune the separation range.
• Tris-Acetate  Gels exhibit an excellent resolution of high–molecular-weight proteins but show poor performance for smaller proteins. Especially large proteins transfer efficiently and evenly to the membrane in western blot approaches.

Conclusion

Selecting a gel system involves matching gel percentage (or gradient) to the target size range and choosing a buffer that maximizes resolution, stability, and compatibility with downstream applications.

Pitfalls

The major problems occur for extremely large or extremely small proteins. Choosing a suboptimal system will lead to poor resolution and/or separation and transfer of the proteins to the membrane.
Overloading the gel will lead to poor resolution and incomplete transfer to the membrane. The membrane itself will also be overloaded.