Non-tagged protein purification overview

Why purify non-tagged proteins?

Although protein tagging enables simple, effective affinity purification of a wide variety of proteins, there are a number of reasons why it is not necessarily the best method for every protein:

• Tags cannot be used to purify endogenous proteins; they can only be used to purify proteins that have been genetically manipulated, which may not be possible or desirable

• In some cases, a tag may interfere with the structure or biological activity of a target protein, or may be difficult to remove without altering its structure or function
• Protein tagging may be unnecessary for proteins with endogenous post-translational modifications or affinity binding sites, such as phosphoproteins, glycoproteins, or antibodies, which can bind specifically to alternative affinity resins

Applications

Phosphoprotein enrichment

Phosphoprotein enrichment with a unique IMAC (immobilized metal affinity chromatography) resin provides an effective affinity-based procedure for isolating phosphorylated proteins from mammalian cells and tissues, using gravity columns or magnetic beads. A similar IMAC resin-based method is available for isolating phosphopeptides, using spin columns or magnetic beads.

• Phosphoproteins isolated using gravity columns are ideal for use in many downstream applications, including mass spectrometry and 2D-PAGE
• Spin column enrichment can enhance HPLC or mass spectroscopy detection of phosphopeptides that would otherwise be undetectable
• Magnetic beads provide microscale phosphoprotein or phosphopeptide enrichment

Glycoprotein enrichment

Glycoprotein enrichment with a boronic acid-based resin provides quick, efficient, and specific enrichment of glycoproteins from complex samples such as human serum, urine, and spinal fluid, using either simple gravity-flow columns or medium-pressure methods such as FPLC. The resin may be used to:

• Specifically enrich low-abundance glycoproteins and reduce sample complexity to facilitate proteomics studies and downstream analysis using mass spectroscopy
• Remove saline contaminants that can interfere with ionization in mass spectroscopy

Antibody purification

Antibody purification with a thiophilic resin is a simple, powerful, and economical alternative to Protein A purification. The resin may be used to purify:

• Common immunoglobulins, including IgG, IgM, IgA, Fab and Fc fragments, and C3 and C4 complement factors
• Single-chain antibodies (e.g., IgE, IgY, and IgM), which cannot be purified using Protein A