FLAG purification

Protein purification of recombinant proteins using affinity chromatography

With proven utility in numerous applications, FLAG® tags enable superior detection and purification of recombinant fusion proteins using anti-FLAG® antibodies
The FLAG® and 3x FLAG systems are useful in protein expression, protein purification, Western blotting, immunocytochemistry, immunoprecipitation, flow cytometry, and pull-down assays.

FLAG® fusion tag cloning and expression vectors

Recombinant protein production has been improved by advances in fusion protein technology. While the original FLAG® peptide (tag sequence: DYKDDDDK) allows sensitive detection, the 3xFLAG™ tag provides enhanced detection. Our portfolio includes vectors for both bacterial and mammalian systems incorporating T7, Tac, or CMV promoters using SnapFast™ expression vectors.

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Anti-FLAG® agarose affinity gels

Agarose affinity gels enable fusion-tagged protein purification using anti-FLAG® antibodies cross-linked to beaded agarose. Good for small to large scale purification, these resins are suitable for gravity flow column purification, low-speed centrifugation in batch processing, and low-pressure chromatography procedures as the beads and gels are designed for lower pressures. EZview affinity gels incorporate a red dye that allows the user to easily differentiate pellet from supernatant, resulting in less protein loss during batch purification. Immunoprecipitation kits include affinity gels, mini-spin columns, and lysis reagent and are specially designed to allow maximal recovery of proteins from immunoprecipitates. • Anti-FLAG® M2 Agarose Affinity Gel uses a mouse monoclonal antibody that recognizes FLAG® sequences at the N-terminal, Met-N-terminal, C-terminal, and internal locations of fusion proteins. Bound antigens can be eluted using competitive FLAG® peptides or reduced pH buffers. • Anti-FLAG® M1 Agarose Affinity Gel uses a purified mouse IgG2B monoclonal antibody that recognizes free N-terminal FLAG fusion proteins. Since binding is Ca2+-dependent, proteins can be eluted with a buffer containing EDTA or by standard methods using FLAG peptide or glycine-HCl buffer (pH 3).

Anti-FLAG® M2 magnetic beads

Anti-FLAG® M2 Magnetic Beads are 4% agarose beads bound to the anti-FLAG® M2 antibody. Magnetic beads are used to purify recombinant fusion-tagged protein using a magnetic rack or platform to separate beads from wash and elution fractions. This procedure allows for rapid processing within seconds and is suitable for low throughput using single microfuge tubes or higher throughput using 96-well plates and automation.

FLAG® purification kits

• The FLAG® M2 Purification Kit utilizes CelLytic Reagents for rapid and efficient cell lysis and protein extraction and anti-FLAG® M2 affinity gel for purification of active FLAG fusion proteins. • The FLAG® HA Tandem Affinity Purification Kit is designed for the isolation of high purity FLAG-HA dual-tagged fusion proteins from complex matrices such as cell lysates and tissue homogenates. The kit is particularly suitable for isolation of protein complexes using TAP (Tandem Affinity Purification) technology.

Anti-FLAG® High Sensitivity M2-coated 96-well plates

Anti-FLAG® High Sensitivity M2-coated 96-well plates are suitable for expression screening, the study of protein-protein interactions, and ELISA assays.

Anti-FLAG® antibodies

FLAG® and 3xFLAG tags include binding sites for highly specific anti-FLAG® monoclonal antibodies, as well as polyclonal antibodies and conjugates. Three monoclonal anti-FLAG® antibodies (M1, M2, and M5) have been produced for the FLAG® tag sequence, each with different epitope binding requirements. Anti-FLAG® antibodies conjugated to alkaline phosphatase and peroxidase are available for ELISA and blotting applications, or labeled with Cy3 or FITC dye for immunofluorescence, immunocytochemistry, immunohistochemistry (IHC), blotting, and flow cytometry.

  • The M2 anti-FLAG® antibody binds N-terminal FLAG®, C-terminal FLAG®, and Met-FLAG® fusion proteins. The universal nature of this antibody renders it more generally applicable to a wide variety of applications.
  • The M1 anti-FLAG® antibody binding is calcium-dependent and specific for the N-terminus of the FLAG® tag. The M1 anti-FLAG® antibody cannot be used with Met-FLAG® (N-terminus preceded by a methionine residue) or C-terminal FLAG® fusion proteins.
  • While M5 anti-FLAG® antibodies can be used with both types of amino-terminal FLAG® fusion proteins, they exhibit a higher affinity for Met-FLAG® fusion proteins. Due to this affinity, M5 anti-FLAG® antibodies are typically used with cytoplasmically expressed FLAG® fusion proteins.

Peptides and control proteins

  • 3X FLAG Peptide is a synthetic 23 amino acid residue peptide containing the Asp-Tyr-Lys-Xaa-Xaa-Asp motif repeated three times. 3X FLAG Peptide is used for competitive elution of 3X FLAGfusion proteins from anti-FLAG® M1 and M2 affinity resins or antibodies.
  • FLAG® Peptide is used for the gentle, competitive elution of N-terminal, Met-N-terminal, or C-terminal FLAG® fusion proteins from anti-FLAG® M1 and M2 affinity resins or antibodies.
  • Produced from an E. coli bacterial alkaline phosphatase fusion protein system, FLAG® control proteins can be used to assure the functional integrity of anti-FLAG® tag antibodies in immunological procedures or as functional controls in immunoaffinity chromatography.