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Protein Purification

Protein Purification

Affinity tagging has revolutionized protein purification by allowing generic purification approaches, and today many proteins can be purified very easily and efficiently.

In research laboratories, protein purification is performed in scales from micrograms and milligrams, while bioprocessing at industry scale can produce kilograms or even tons. Preparative purification of proteins commonly involves chromatography, and GE Healthcare offers a large range of chromatography media to purify proteins using manual methods or automated chromatography systems. Our tools cover a variety of prepacked formats for convenient protein purification such as gravity columns, spin columns, 96-well plates, as well as prepacked columns for use with automated chromatography systems.

These pages will give you an overview of protein purification strategies and tools you can use for purifying tagged and untagged proteins. For step-by-step protocols and practical tips about protein purification, download one of our in-depth handbooks or watch one of our protein purification videos.

If you are teaching protein purification, you can find helpful resources in our protein purification education site.

Antibodies His Tag Strep-tag™ II MBP Tag GST Tag Untagged

Antibody Purification

We offer tools for the purification of monoclonal and polyclonal antibodies from different sources. The basis for antibody affinity purification is the high affinity and specificity of protein G and protein A for the Fc region of IgG from a variety of species. We offer chromatography media with protein G and protein A immobilized to several different matrices resulting in an excellent means of isolating IgG and IgG subclasses from ascites, cell culture supernatants, and serum.

We also offer chromatography media with recombinant protein L ligand for purification of antibodies and antibody fragments. Protein L has a strong affinity to the variable region of kappa light chains and can be used to capture a wide range of antibody fragments such as Fab fragments, single-chain variable fragments (scFv), and domain antibodies (Dabs).

Learn more about our products for antibody affinity chromatography.

Histidine-Tagged Protein Purification

Histidine tags are widely used because they are small and rarely interfere with the function, activity, or structure of target proteins. Immobilized metal ion affinity chromatography (IMAC) is the most common method for purifying histidine-tagged proteins. IMAC chromatography media charged with divalent metal ions such as nickel or cobalt selectively retain histidine-tagged proteins and allow for the purification of insoluble histidine-tagged proteins from inclusion bodies when denaturing conditions are used. Successful IMAC purification gives a high yield of pure and active target protein.

Since many proteins have intrinsic histidine and/or cysteine amino acid residues, other nonspecific proteins can bind to the IMAC media together with the target protein. In such cases, it is often necessary to optimize binding, wash, and elution conditions by varying the concentration of imidazole in these solutions. Increasing the concentration of imidazole in the binding and wash buffers generally decreases nonspecific binding, whereas lower concentrations give stronger affinity interaction. The key is finding the right balance.

Learn more about our products for histidine-tagged protein purification.

Strep-tagTM II Tagged Protein Purification

Strep-tagTM II is a small tag of only eight amino acids with a relative molecular mass (Mr) of just 1000. Its small size is very beneficial, since in most cases it does not interfere with structural and functional studies, and therefore does not have to be removed.

Strep-tagTM II binds specifically to StrepTactinTM Sepharose High Performance, which has Strep-TactinTM ligand immobilized on a Sepharose base matrix to yield pure target protein. The binding affinity of Strep-tagTM II to the immobilized ligand is nearly 100-fold greater than to streptavidin, making suitable for purifying Strep-tagTM II proteins. Purification is run under physiological conditions, and mild elution with desthiobiotin preserves the activity of the target protein.

Learn more about Strep-tag II tagged protein purification.

MBP-Tagged Protein Purification

Maltose binding protein (MBP) is a useful affinity tag that can increase the expression level and solubility of the resulting tagged protein. The MBP tag also promotes proper folding of the attached protein. Since MBP increases solubility, the tag is particularly useful for recombinant proteins that accumulate in an insoluble form (inclusion bodies).

Affinity purification takes place under physiological conditions and mild elution is performed using maltose. The mild elution preserves the activity of the MBP-tagged protein.

Learn more about our products for MBP-tagged protein purification.

GST-Tagged Protein Purification

Glutathione S-transferase (GST) Gene Fustion System is a versatile system for the expression, purification, and detection of GST-tagged proteins produced in E. coli. Purification of GST-tagged proteins can be performed under very mild conditions, which preserves the function and antigenicity of the target protein.

The GST tag may also increase the solubility and stability of the target protein. The GST tag is large with a relative molecular mass (Mr) of 26000. A specific cleavage sequence allows simple removal of the GST tag with the use of different proteases after purification.

Typical features of GST-tagged proteins include high binding specificity to glutathione ligands on Glutathione Sepharose chromatography media, resulting in greater than 90% purity in one step of the eluted target molecule.

Learn more about our products for GST-tagged protein purification.

Untagged or Native Protein Purification

Untagged recombinant and native proteins usually require a multi-step purification protocol to obtain sufficient purity. These proteins may come from natural sources or have been overexpressed without a tag because the presence of a tag would interfere with the use of the protein.

The purification strategy Capture, Intermediate Purification and Polishing (CIPP) is a tool to simplify planning and execution of protein purification. The strategy gives guidelines for how to combine purification methods in the best way to reach the set goals.

Learn more about CIPP  on the Protein Purification Strategies page.

We also offer specialized chromatography media that have pre-immobilized ligands that bind specific classes of molecules such as serine proteases, S-transferases, and calmodulin-requiring enzymes. Learn more about our range of group-specific chromatography media.

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