After successful expression of a recombinant protein, it is often desirable to purify the protein to permit its characterization or its use in various applications. There are many strategies that can be applied to purify proteins and entire books have been written on this subject. However, one type of purification that is commonly used by researchers is affinity chromatography. This method is often preferred because it can rapidly yield small but useful quantities of pure recombinant protein in a single chromatography step. In its most typical application, a protein or peptide “tag” that has affinity for a specific immobilized substrate is appended to the target recombinant during its expression. The tagged protein can then be recovered from a complex mixture (such as a cell lysate) by contacting the mixture with a chromatography resin that displays a substrate to which the tag selectively binds. Common tags include polyhistidine sequences (“His-tags”) that specifically bind to resins displaying nickel or cobalt ions, peptide epitopes that interact with specific immobilized antibodies, or protein domains (e.g. maltose binding protein or chitin binding domain) that bind to chromatography resins that display a specific sugar. Chromatography resins may be used in several formats including packed columns or spin columns. Additionally, substrates or proteins that specifically bind to tags may be immobilized to magnetic beads to permit isolation of tagged proteins upon their exposure to a magnetic field.
FAQs for Affinity Purification
Protocols for Affinity Purification
- Cleavage of the Fusion Protein Generated Using The pMAL Protein Fusion and Purification System (E8200)
- Cloning a PCR Fragment Into a pMAL Expression Vector (E8200)
- Cross-linking of IgG to Protein A or G Beads
- Goat Anti-Mouse IgG Magnetic Beads Protocol
- Goat Anti-Rabbit IgG Magnetic Beads Protocol
- Goat Anti-Rat IgG Magnetic Beads (S1433)
- Immunoprecipitation using Protein A/G Magnetic Beads
- Isolation of CBD-fusion protein using Chitin Magnetic Beads
- Isolation of MBP-fusion protein using Amylose Magnetic Beads
- Isolation of MBP-fusion protein using Anti-MBP Magnetic Beads
- mRNA Isolation using Streptavidin Magnetic Beads (NEB #S1420 and NEB #S1421)
- Phage Display: Solution-phase Panning with Affinity Bead Capture
- Purification of a Fusion Protein generated by The pMAL Protein Fusion and Purification System (E8200)
- Purification of IgG using Protein A/G Magnetic Beads
- Quick Start Guide (E8200)
- Separating the Protein of Interest from MBP after Protease Cleavage Using The pMAL Protein Fusion and Purification System (E8200)
- Use SNAP-Capture Magnetic Beads (S9145)
Protein Expression & Purification Brochure
The Protein Expression and Purification brochure provides information on the advanced tools for protein expression and purification offered by NEB.
- Affinity of Protein A/G for IgG Types from Different Species
Other Tools & Resources
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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.