Achieve High Specificity and Sensitivity in Protein Research with FLAG® Peptide Tags
Epitope tags play a crucial role in life sciences, facilitating the expression and purification of recombinant proteins in biotechnology and allowing researchers to examine the interactions of molecules in biological systems. Among these tags, FLAG® (DYKDDDDK) stands out – it has a small size and hydrophilic properties, which reduce interference with protein functionality, increasing specificity and sensitivity. Read more to learn about the benefits and use of these products in protein research.
Section overview
Key Benefits of the FLAG® Product Portfolio
FLAG® tags are compatible with various techniques, including Western blotting and immunoprecipitation, immunohistochemistry, and cell sorting. The FLAG® tag is one of the most reliable epitope tags in molecular biology research, reflected in thousands of primary literature citations. FLAG® products provide high specificity, sensitivity, binding capacity, and efficient elution that researchers need.
- Best in Class Specificity: Our ANTI-FLAG® resins achieve the highest specificity compared to competitors for purification and immunoprecipitation, bringing higher confidence and reproducibility to your research.
- Enhanced Sensitivity of FLAG® Antibodies: Our FLAG® antibodies demonstrate superior sensitivity (2 ng guaranteed, as low as 0.1 ng observed) among all major competitors resulting in smaller sample sizes needed for your results.
- High Binding Capacity and Efficient Elution: Optimized binding affinity balances specific binding with gentle elution options. Users can expect capacities of greater than 0.6 mg/mL and workflows typically requiring 2 hours or less.
Superior Specificity for Immunoprecipitation and Purification with ANTI-FLAG® Resins
FLAG®-tagged proteins can be purified from various expression systems using ANTI-FLAG® M2 affinity gels or ANTI-FLAG® M2 magnetic beads with best-in-class specificity. Our scientists used the cell systems E. coli, P. pastoris, CHO, and SF9 to examine FLAG® antibody specificity in a complex molecular environment.
To measure antibody specificity, ANTI-FLAG® resins were introduced to capture FLAG®-tagged bacterial alkaline phosphatase spiked into cell lysate. After washing away non-specific contaminants, all the bound proteins were eluted with low pH glycine for analysis using two workflows: 1) trypsin digestion followed by LC-MS for identification and quantification of all eluted species; 2) SDS-PAGE. Below are examples of the percent purity after elution with Non-Magnetic Resin for CHO (Figure 1), E.coli (Figure 2), P.pastoris (Figure 3), and SF9 (Figure 4) lysates; and with Magnetic Resin for CHO (Figure 5), E.coli (Figure 6), P.pastoris (Figure 7), and SF9 (Figure 8) lysates.
Figure 1. Comparison of FLAG® Products Versus Competitors for Non-Magnetic Resin Specificity Measured by LC-MS and SDS-PAGE for Proteins Purified from CHO lysates.SDS-PAGE gel (A) and bar graph (B) showing the percent purity of eluted N-FLAG-BAP™ from ANTI-FLAG® M2 Agarose A2220 (Lane 1, Blue), Competitor G (Lane 2, green), and Competitor T (Lane 3, pink) for lysates from CHO. Note: SDS-PAGE sensitivity is significantly lower than LC-MS peptide mapping.
Figure 2. Comparison of FLAG® Products Versus Competitors for Non-Magnetic Resin Specificity Measured by LC-MS and SDS-PAGE for Proteins Purified from E. coli lysates.SDS-PAGE gel (A) and bar graph (B) showing the percent purity of eluted N-FLAG-BAP™ from ANTI-FLAG® M2 Agarose A2220 (Lane 1, blue), Competitor G (Lane 2, green), and Competitor T (Lane 3, pink) for lysates from E.coli. Note: SDS-PAGE sensitivity is significantly lower than LC-MS peptide mapping.
Figure 3. Comparison of FLAG® Products Versus Competitors for Non-Magnetic Resin Specificity Measured by LC-MS and SDS-PAGE for Proteins Purified from P.pastoris lysates.SDS-PAGE gel (A) and bar graphs (B) showing the percent purity of eluted N-FLAG-BAP™ from ANTI-FLAG® M2 Agarose A2220 (Lane 1, blue), Competitor G (Lane 2, green), and Competitor T (Lane 3, pink) for lysates from P.pastoris. Note: SDS-PAGE sensitivity is significantly lower than LC-MS peptide mapping.
Figure 4. Comparison of FLAG® Products Versus Competitors for Non-Magnetic Resin Specificity Measured by LC-MS and SDS-PAGE for Proteins Purified from SF9 lysates.SDS-PAGE gel (A) and bar graph (B) showing the percent purity of eluted N-FLAG-BAP™ from ANTI-FLAG® M2 Agarose A2220 (Lane 1, blue), Competitor G (Lane 2, green), and Competitor T (Lane 3, pink) for lysates from SF9. Note: SDS-PAGE sensitivity is significantly lower than LC-MS peptide mapping.
Figure 5. Comparison of FLAG® Products Versus Competitors for Magnetic Resin Specificity Measured by LC-MS and SDS-PAGE for Proteins Purified from CHO Lysates.SDS-PAGE gel (A) and bar graph (B) showing the percent purity of eluted N-FLAG-BAP™ from ANTI-FLAG® M2 Agarose M8823 (Lane 1, purple), Competitor G (Lane 2, green), and Competitor T (Lane 3, pink) for lysates from CHO. Note: SDS-PAGE sensitivity is significantly lower than LC-MS peptide mapping.
Figure 6. Comparison of FLAG® Products Versus Competitors for Magnetic Resin Specificity Measured by LC-MS and SDS-PAGE for Proteins Purified from E.coli Lysates.SDS-PAGE gel (A) and bar graph (B) showing the percent purity of eluted N-FLAG-BAP™ from ANTI-FLAG® M2 Agarose M8823 (Lane 1, purple), Competitor G (Lane 2, green), and Competitor T (Lane 3, pink) for lysates from E.coli. Note: SDS-PAGE sensitivity is significantly lower than LC-MS peptide mapping.
Figure 7. Comparison of FLAG® Products Versus Competitors for Magnetic Resin Specificity Measured by LC-MS and SDS-PAGE for Proteins Purified from P.pastoris lysates. SDS-PAGE gel (A) and bar graph (B) showing the percent purity of eluted N-FLAG-BAP™ from ANTI-FLAG® M2 Agarose M8823 (Lane 1, purple), Competitor G (Lane 2, green), and Competitor T (Lane 3, pink) for lysates from P.pastoris. Note: SDS-PAGE sensitivity is significantly lower than LC-MS peptide mapping.
Figure 8. Comparison of FLAG® Products Versus Competitors for Magnetic Resin Specificity Measured by LC-MS and SDS-PAGE for Proteins Purified from SF9 lysates.SDS-PAGE gel (A) and bar graph (B) showing the percent purity of eluted N-FLAG-BAP™ from ANTI-FLAG® M2 Agarose M8823 (Lane 1, purple), Competitor G (Lane 2, green), and Competitor T (Lane 3, pink) for lysates from SF9. Note: SDS-PAGE sensitivity is significantly lower than LC-MS peptide mapping.
Our FLAG® products offer superior specificity compared to competitors. Eliminating non-specific interactions improves confidence and reproducibility. |
Enhanced Sensitivity with FLAG® Antibody Products
Our FLAG® antibody products demonstrate enhanced sensitivity compared to competitor antibodies, allowing for protein detection at lower quantities. To evaluate this, our scientists performed a dot-blot assay comparing ANTI-FLAG® antibodies, M2 Mouse Monoclonal Antibody (Cat. No. F1804 and Cat. No. F3165), and competitor antibodies. Successively smaller quantities of FLAG®-tagged protein were applied to a nitrocellulose membrane, then the spots were treated with a 1 μg/mL antibody solution and imaged via a secondary antibody conjugated to horseradish peroxidase (Figure 9).
Figure 9. Sensitivity Comparison of ANTI-FLAG® Antibodies to Competitors. Dot blot (A) and corresponding bar graph (B) showing luminescence intensity for the quantity of FLAG®-tagged bacterial alkaline phosphatase (BAP) in dot (ng) treated with ANTI-FLAG® M2 mouse monoclonal, Cat No. F1804 (cyan) and Cat No. F3164 (purple), Competitor G (green), and Competitor T (pink) Antibody solutions. Bar graphs include error bars from 4 replicates.
Our FLAG® antibodies offer enhanced specificity compared to competitor antibodies.
Elution Strategies: Balancing Binding Capacity and Elution Efficiency with 3XFLAG™ peptide
Efficient elution of FLAG® fusion proteins is possible using a variety of methods. These include competitive elution with 3xFLAG™ peptide, pH based acidic elution, and heating with SDS and a reducing agent (Figure 10). Binding kinetics are finely tuned for both binding and elution, ensuring optimal yields and reproducible results.
Figure 10. Comparison of the Three Methods of Elution used with ANTI-FLAG® M2 Magnetic Beads.Three protein gels showing the purity of N-terminal (A), C-terminal (B) and A431 spiked with N-terminal (C) control proteins comparing starting material (St, Lane 1), after elution with 3XFLAG™ peptide (3X, Lane 2), after elution with 2.5 glycine (pH, Lane 3), and after elution with sodium dodecyl sulfate and reducing agent (SDS, Lane 4).