Size Exclusion Chromatography (SEC) remains a cornerstone method for protein purification, valued for its ability to deliver high-purity samples with minimal loss of biological activity. From structural analysis to functional studies and therapeutic development, the purity of protein preparations directly impacts experimental outcomes.
It’s a simple, baseline reality: Producing high-quality protein samples is critical to accurate and reproducible scientific research. Poor-quality protein samples yield inconsistent experimental results, making interpreting and replicating data difficult. Ultimately, a poor sample undermines scientific research.
Principles of the SEC
Size Exclusion Chromatography, or gel filtration chromatography, separates proteins based on their molecular size as they pass through a column filled with porous beads. Proteins navigate through the column based on size: larger molecules elute faster as they bypass the pores, while smaller proteins enter the pores and take a longer path to elute.
Size exclusion chromatography uses resins composed of agarose, polyacrylamide, or dextran polymer beads. Unlike other chromatographic resins, size exclusion resins do not directly bind target molecules. Separation of biomolecules occurs when particles of varying sizes are included or excluded from the pores in the matrix. Purifying proteins involves removing unwanted contaminants like nucleic acids, lipids, and other proteins. SEC is a critical final step to ensure the target protein isolates in its pure, biologically active form. SEC stationary phases have specified fractionation ranges that outline the molecular weight ranges that a given matrix can separate. Small biomolecules, like peptides, need size exclusion resins with low fractionation ranges. Larger particles, like antibodies, require stationary phases with higher fractionation ranges.
Key Benefits That Make the SEC Indispensable
SEC provides the native state of proteins in high resolution, allowing researchers to distinguish between closely related species, such as monomers and aggregates. SEC's flexibility enables its use across a broad range of molecular weights, from small peptides to large complexes, allowing it to be scaled from small analytical applications to large preparative purifications without compromising performance.
Due to its reliability, versatility, and non-denaturing separation process, SEC is one of the cornerstone techniques in protein research, making it an ideal choice where high purity and intact protein structure are required. For example, in:
SEC enables the researchers to rely on critical tasks such as:
Best Practices for Successful Size Exclusion Chromatography
To ensure accurate results while using SEC, researchers must focus on the following parameters:
G-Biosciences offers the following products to enhance SEC workflows. As protein research evolves, robust purification methods are more important than ever
Figure 1: Size exclusion column:
Protein Purification Handbook
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