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What is an IgG-Fc-tag or IgG-Fc Chimeric?
IgG-Fc tag is the constant region (domain 3 and 4) of immunoglobulin heavy-chain. It is fused to the C-terminus of a protein and hence it recembles a mouse/human chimeric antibody in a way, and sometimes the Fc-fusion protein is also called Fc chimeric protein. The Fc-tag is about 25 KDa.
Why use an IgG-Fc-tag for protein expression and production?
Adding an IgG-Fc tag to a protein not only allows rapid and simple detection of protein expression by commercially available ELISA kit, but also assist simple affinity purification of the Fc-tagged protein by protein A, protein G, and protein L affinity purification resins. Furthermore, adding an IgG-Fc tag to a protein often increases protein expression yield.
Why IgG-Fc-tagged Protein shows as a Dimer on non-reduced SDS Page?
Because IgG antibody Heavy chain forms a dimer naturally through the hinge regions cysteines residuals, the IgG-Fc chimeric protein always forms a disulfide-bonded dimer. It shows as a monomer on reduced SDS page, but shows as a homo-dimer on a non-reduced SDS page.
What choices of IgG-Fc can I choose for my Fc-chimeric protein?
You can choose the Fc region of any IgG antibodies from multiple species, depending on your preference of application. Human IgG1-Fc is the most widely used Fc-tag due to easy detection and high binding affinity to protein A resin.
An incomplete list of IgG-Fc for Fc tag is listed below for your reference:
Human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc
Mouse IgG1-Fc, IgG2a-Fc, IgG2b-Fc, IgG3-Fc
Rat IgG1-Fc, IgG2a-Fc, IgG2b-Fc, IgG2c-Fc
How to Purify Fc-tagged Proteins?
Fc-tagged recombinant protein can be affinity purified directly from a cell culture lysate or supernatant. Depending on the selection of the IgG species and the subtype, the Fc-tagged protein binds to the protein A, protein G, or protein L affinity purification resins just like an IgG antibody. Ater washing away residual impurities, bound Fc-tag proteins can be eluted off the affnity column by low pH buffer. For more information on our high quality and low cost antibody affinity purification resins, please refer to: Protein A Affinity Resin, Protein G Affinity Resin, Protein L Affinity Resin .
How do I cleave of the IgG-Fc-tag after purification?
In some applications, it is desirable to remove the Fc-tag, for example, for protein crystalization. To allow cleavage of the Fc-tag, a protease cleavage site needs to be engineered between the tag and the protein. An EK cleavage site behind the Fc-tag (Fc-EK site-protein structure) can allow complete removal of the Fc-tag and the cleavage site, leaving no additional amino acids after the specific cleavage of the Fc-tag. For more information on the cleavage site and tag removal by EK and HRV-3C protease, please refer to: Enterokinase (EK), HRV-3C (human rhinovirus protease).