FLAG-tag Protein Expression, Production, and Purification Basics

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What is a FLAG-tag?

A FLAG-tag, or FLAG octapeptide, is a peptide sequence of DYKDDDDK, which can be fused tgo either the N- or C-terminus of a protein to facilitate detection and purification by a FLAG-tag specific monoclonal antibody.

Why use a FLAG-tag for protein expression and production?

Adding a FLAG-tag to the N- or C-terminus of a protein allows rapid purification of the over-expressed recombinant protein, by using a FLAG-tag specific monoclonal antibody conjugated to agarose beads. Previsouly, commmercial FLAG-tag affinity purification resin is very expensive and can only be used for a couple of times. After many years of development, Sino Biological has developed an excellent FLAG-tag specific monoclonal antibody that can be used for various detection applications as well as be conjugated to agarose for affinity purification. The improved FLAG-tag affinity resin is affordable and can tolerate low pH elution, which increase the life-span of the resin.

In addition, antibodies specific to the FLAG-tag can be used to detect the tagged protein expression level in cell culture, by ELISA and western blot, etc.

What is the Molecular Wieght of the FLAG-tag?

The molecular wieght of the DYKDDDDK-tag (FLAG-tag) is 1012 Da.

How do I cleave of the FLAG-tag after purification?

In some applications, it is desirable to remove the FLAG-tag, for example, for protein crystalization. To allow cleavage of the FLAG-tag, a protease cleavage site needs to be engineered between the tag and the protein. An EK cleavage site behind the FLAG-tag (FLAG-EK site-protein structure) can allow complete removal of the FLAG-tag and the cleavage site, leaving no additional amino acids after the specific cleavage of the FLAG-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).

How to Purify FLAG-tagged Proteins?

FLAG-tagged recombinant protein can be affinity purified directly from a cell culture lysate or supernatant. The FLAG-tagged protein binds to the FLAG-tag specific monoclonal antibody conjugated on an agarose gel. After washing away residual impurities, bound FLAG-tag proteins can be eluted off the affnity column by high concentration of the FLAG-tag peptide or by low pH buffer. For more information on usage of the FLAG-tag affinity purification resin, please refer to: FLAG-tag Affinity Resin.

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