How do you elute a flag tag?

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 affinity column by high concentration of the FLAG-tag peptide or by low pH buffer.

How do you elute flag tagged protein?

1. Elution of the FLAG fusion protein may be achieved under native conditions by competition using 3X FLAG peptide (F4799). This elution is the most efficient method.
2. Elution may be performed under acidic conditions using 0.1 M glycine HCl, pH 3.5.
3. Elution may be achieved by electrophoresis using SDS-PAGE sample buffer.

What is Flag tag sequence?

FLAG-tag, or FLAG octapeptide, or FLAG epitope, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology, having the sequence motif DYKDDDDK (where D=aspartic acid, Y=tyrosine, and K=lysine). A FLAG-tag can be used in many different assays that require recognition by an antibody.

How does his-tag purification work?

His-tag purification uses the purification technique of immobilized metal affinity chromatography, or IMAC. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid.

How do I purify GST tagged protein?

Glutathione Sepharose resins are often used for purification. The binding of a GST-tagged protein to the ligand is reversible, and the protein can be eluted by adding reduced glutathione to the elution buffer. Optional removal of the GST tag can be performed on the column or after elution.

Is flag an epitope?

FLAG tag (DYKDDDDK) is an epitope tag widely adopted for its high specificity and purity in protein purification. Anecdotally, it is commonly used as a follow-on or alternative to His tag when high purity is desired.

What is a 3X FLAG tag?

General description. The 3X FLAG Peptide is a synthetic peptide of 23 amino acid residue. The Asp-Tyr-Lys-Xaa-Xaa-Asp motif is repeated three times in the peptide. Eight amino acids at the C-terminus make up the classic FLAG sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys).

What are purification tags?

Protein tags are most frequently used to purify proteins for which no protein-specific antibody exists. Such tags include his (polyhistidine), FLAG (DYKDDDDK), GST, and Myc tags, which are fused to proteins of interest using expression vector systems.

How does NI-NTA purification work?

The Ni-NTA Purification System is designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and mammalian cells. The system is designed around the high affinity and selectivity of Ni-NTA Agarose for recombinant fusion proteins that are tagged with six tandem histidine residues.

How do you elute protein from GST beads?

To elute the protein from the GST tag and agarose bead, add 10µl of thrombin (10 units) per mg GST tagged protein. 2. Mix gently and incubate at room temperature for 2-16 hours.

What is FLAG-tag purification?

FLAG-tag is one of the commonly used purification technologies for recombinant proteins. An antibody, M2, specifically binds to the FLAG-tag whether it is attached to N- or C-terminus of proteins to be purified. The bound proteins are generally eluted by competition with a large excess of free FLAG peptide.

Can EDTA be used in affinity purification of flag™ fusion proteins?

In order to extend the application area of the FLAG™ tag, another anti-Flag monoclonal antibody (anti-Flag M2) for use in affinity purification of FLAG™ fusion proteins was raised. The binding of the anti-Flag M2 antibody is not calcium-dependent, therefore, bound antigens cannot be eluted from the affinity column by chelating agents, such as EDTA.

What is the molecular weight of a FLAG tag?

FLAG-tag, or FLAG octapeptide, or FLAG epitope, is the first epitope tag designed for fusion proteins and is the only patented tag. The molecular weight of the DYKDDDDK-tag (FLAG-tag) is 1012 Da. The multiple polyanionic amino acids in the FLAG tag are less likely to affect the activity of a target protein.

How are flag-tagged proteins eluted off the affinity column?

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 affinity column by high concentration of the FLAG-tag peptide or by low pH buffer.