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Protein Expression & Purification


Products >  Protein_Expression_and_Purification >  Protein_Learning_Center >  Tagged_Protein_Purification >  FLAG-Tagged_Protein_Purification_Overview

FLAG-Tagged Protein Purification Overview

Technologies

Takara Clontech offers two different technologies for FLAG-tagged protein purification:

Agarose Beads

Magnetic
Beads

Introduction

The FLAG (DYKDDDDK epitope) tag is a small, widely used, well-characterized hydrophilic peptide tag (8 amino acids long). It binds to the Anti-DYKDDDDK Antibody and can be incorporated on either the N- or C-terminus, and expressed in bacterial, yeast, mammalian, and baculovirus-infected insect cells.

Applications

The FLAG tag may be used for the following applications:

  • When a small tag is required to minimize its effect on protein function
  • When a hydrophilic tag is required
  • When a low metabolic load is needed to maintain normal cell physiology and/or to increase protein expression
  • When highly specific detection is necessary
  • To monitor recombinant protein expression in bacterial, yeast, mammalian, or insect cells
  • For co-immunoprecipitation studies, Western blots, and flow cytometry
  • To monitor subcellular localization of tagged proteins in mammalian cells

Purifying FLAG-Tagged Proteins Using Immunoprecipitation

Immunoprecipitation (IP) is a technique for separating a protein of interest from a solution containing thousands of different proteins, such as a cell lysate, by precipitating it from the solution using an antibody that binds specifically to that protein. Proteins that contain peptide tags, such as the FLAG (DYKDDDDK epitope) tag, can be isolated and concentrated using an antibody specific for this tag, provided that the antibody is attached to a solid substrate. A tag-specific antibody can also be used to precipitate protein complexes, in a process known as co-immunoprecipitation (Co-IP). Takara Clontech offers a choice of anti-DYKDDDDK antibodies attached to two different substrates, agarose beads or magnetic beads, for your IP and Co-IP experiments.

The Magnetic DYKDDDDK Immunoprecipitation Kit offers a simple, rapid method for purifying or immunoprecipitating FLAG-tagged fusion proteins from cell lysates—using magnetic beads cross-linked to a monoclonal anti-DYKDDDDK antibody (which recognizes the FLAG epitope). The kit also includes the Magnetic Beads Immunoprecipitation Buffer Set (optimized to provide extremely low background) and the ProteoGuard Protease Inhibitor Cocktail (specially designed to inhibit endogenous proteases released during cell lysis).

Immunoprecipitation of AcGFP1-DYKDDDDK from bacterial and mammalian cell lysates using the Magnetic DYKDDDDK Immunoprecipitation Kit. After lysis using standard protocols, cell lysates from E. coli cells (Panel A) and HeLa cells (Panel B), each expressing AcGFP1-DYKDDDDK, were immunoprecipitated using the Magnetic DYKDDDDK Immunoprecipitation Kit protocol. The lysates and final eluates were analyzed using SDS-PAGE and silver staining. Lanes 1: Lysate. Lanes 2: Eluate. Lanes M: Molecular weight marker.

Anti-DYKDDDDK Beads consist of a monoclonal anti-DYKDDDDK antibody (which recognizes the FLAG epitope), crosslinked to agarose beads. These beads are used to purify or immunoprecipitate DYKDDDDK-tagged or FLAG-tagged fusion proteins from cell lysates. Clontech's Anti-DYKDDDDK Beads and Immunoprecipitation Buffer Set can be purchased separately to make a complete Co-IP kit for purification of FLAG-tagged proteins.

Clontech's Anti-DYKDDDDK Beads for immunoprecipitation provide higher purity and lower background when compared with Competitor S's anti-FLAG beads. A cell lysate was prepared from 293T cells transfected with N-terminally FLAG-tagged AcGFP1, and 100 µl samples of lysate were immunoprecipitated with 25 µl of either Anti-DYKDDDDK Beads or Competitor S's beads in a total volume of 1.1 ml. Immunoprecipitates were washed 4X with PBS, and 5 µl of each final 50 µl sample was analyzed on a Western blot and an SDS-PAGE gel stained with silver stain (shown). Anti-DYKDDDDK Beads were compared to Competitor S's beads at an antibody/bead ratio of 2 mg Ab/ml of beads.

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