In the world of biomedical research, protein purification is more than just a lab technique—it’s a gateway to understanding the inner workings of life. From unraveling disease mechanisms to advancing drug discovery, high-quality protein samples are the foundation for groundbreaking discoveries.
Yet, researchers often face significant roadblocks. Traditional purification methods can be slow, expensive, and inefficient, especially when it comes to complex and low-abundance membrane proteins. These proteins, which play vital roles in cell signaling and are critical for structural biology studies, are notoriously difficult to isolate due to their sensitivity to environmental changes and their scarce expression levels.
A Smarter Solution for Flag-Tagged Proteins
To address these challenges, Yeasen has developed the Anti-Flag Affinity Gel (Cat#20585ES)—a next-generation tool designed for the purification and immunoprecipitation (IP) of Flag-tagged fusion proteins, including secreted and membrane proteins.
What sets this gel apart is its advanced construction:
- High-quality monoclonal antibody: Mouse IgG2b, clone 3F5, ensures strong and specific binding to Flag-tagged proteins.
- Robust matrix: A 4% highly cross-linked agarose gel, covalently coupled to the antibody, provides excellent stability and low background.
- Versatile applications: Ideal for both protein purification and IP workflows, making it a flexible solution for labs tackling diverse protein research.
Why It Matters
For scientists working with challenging proteins, every step saved in purification means more time and energy to focus on analysis and discovery. By combining specificity, efficiency, and reliability, the Anti-Flag Affinity Gel empowers researchers to generate cleaner, higher-yield protein samples with less hassle.
Whether you’re studying signaling pathways, working on structural biology projects, or developing therapeutic proteins, this gel provides the consistency and reproducibility you need to move your research forward.
Key Benefits at a Glance
- High specificity – Built with high-quality mouse IgG2b monoclonal antibody for strong, selective binding to Flag-tagged proteins.
- High loading capacity – Supports up to ≥1.1 mg protein per mL of gel for efficient purification.
- High purity – Minimizes non-specific binding to deliver cleaner Flag-tagged proteins.
- Flexible binding – Compatible with Met-modified N-terminal, N-terminal, and C-terminal Flag fusion proteins.
- Broad applicability – Suitable for both protein purification and immunoprecipitation (IP) workflows.
What truly makes the Yeasen Anti-Flag Affinity Gel (Cat#20585ES) stand out is not only its design but also its performance.
1. Flexible binding&High specificity
M: Protein Marker
1: Flow-through of Flag-C protein (pre-column)
2: Unbound fraction of Flag-C protein (post-column)
3: Eluate (eluted at pH 2.7)
4: Eluate (eluted at pH 12)
5: Eluate (eluted with 1× Flag Peptide)
6: Used gel (after elution at pH 2.7)
7: Used gel (after elution at pH 12)
8: Used gel (after elution with 1× Flag Peptide)
a: Flag-N b: Flag-M c: Flag-C
Figure 1. Purification of low-abundance Flag-tagged proteins using Anti-Flag Affinity Purification Gel
The Anti-Flag Affinity Purification Gel (Cat. #20585ES) specifically captures N-terminal Flag-fusion proteins (Flag-N), N-terminal Flag-fusion proteins with Met modification (Flag-M), and C-terminal Flag-fusion proteins (Flag-C), even when expressed at very low levels. These results demonstrate the high specificity and broad applicability of the gel for purifying diverse Flag-tagged constructs, including secreted and membrane proteins.
2. High purity
M: Protein Marker
1: Flow-through of Flag-C protein (pre-column)
2: Unbound fraction of Flag-C protein (post-column)
3: Eluate (eluted at pH 2.7)
4: Eluate (eluted at pH 12)
5: Eluate (eluted with 1× Flag Peptide)
6: Used gel (after elution at pH 2.7)
7: Used gel (after elution at pH 12)
8: Used gel (after elution with 1× Flag Peptide)
a: Yeasen b: Supplier S* c: Supplier G*
Figure 2. Comparison of low-abundance protein purification using Anti-Flag gels.
Under very low target protein expression conditions, such as membrane proteins, Yeasen’s Anti-Flag Gel (Cat#20585ES) delivered higher yield and purity than other suppliers’ products. Recovery was further improved using Yeasen’s 1× Flag Peptide (Cat#20572ES) for elution.
Troubleshooting & FAQ
|
Problem |
Possible Cause |
Recommended Solution |
|
High backpressure in the column |
Resin clogged |
Clean the resin. |
|
Lysate contains small solid particles |
Filter samples through a 0.22 µm or 0.45 µm filter, or centrifuge before loading. |
|
|
Sample too viscous (high nucleic acid content) |
Extend cell disruption until viscosity decreases, or add DNase I (final conc. 5 µg/mL) with Mg²⁺ (1 mM) and incubate on ice for 10–15 min. |
|
|
Buffer too viscous |
Organic solvents or stabilizers (e.g., glycerol) may cause high backpressure; reduce flow rate. |
|
|
No target protein in elution fractions |
Flag-tagged protein denatured |
Avoid harsh lysis; use gentle lysis conditions. |
|
Elution method not suitable |
Switch to competitive elution using 1× Flag peptide. |
|
|
Target protein aggregates |
Add DTT (final conc. 1–10 mM) before lysis to reduce aggregation. |
|
|
Flag conformation altered in fusion protein |
Test binding affinity; if reduced, adjust construct design or binding conditions. Lower binding temp to 4 °C and wash thoroughly. |
|
|
Incomplete protein elution |
Elution volume too small |
Increase elution volume and slow down the flow rate. |
|
pH of Gly-HCl elution buffer changed |
Prepare fresh elution buffer. |
|
|
Insufficient incubation |
Extend incubation time of elution buffer with resin. |
|
|
Multiple bands on SDS-PAGE or Western blot |
Flag-fusion protein degraded |
Add protease inhibitors (e.g., 1 mM PMSF) to lysis buffer. |
|
Proteolysis during expression |
Use protease-deficient host strains (e.g., lon⁻ or ompT⁻). |
|
|
Over-disruption of cells |
Shorten sonication time; add lysozyme (0.1× culture volume, 10 mg/mL in 25 mM Tris-HCl, pH 8.0) before sonication; avoid foaming. |
|
|
Antibody binding to E. coli proteins |
Remove bound antibodies by sonication; confirm with Western blot. |
A Complete Solution for Flag-Tagged Fusion Proteins
Yeasen doesn’t stop at just one product. We offer a full suite of Flag-tagged protein research tools, designed to work seamlessly together:
- Recombinant Enterokinase (rEK, Cat#20395ES): Efficiently removes Flag tags at the DDDDK site, restoring the protein to its native form for structural and functional studies.
- Anti-Flag Purification Magnetic Beads (Cat#20785ES): Polymer-based agarose magnetic beads (~70 μm) with covalently coupled anti-Flag antibodies, allowing one-step magnetic separation and purification.
- Anti-Flag Immunomagnetic Beads (Cat#20565ES): 200 nm silica-based beads with anti-Flag antibodies, designed for IP and Co-IP applications. For convenience, the Flag-tagged Protein IP/Co-IP Kit (Cat#20765ES) provides everything needed in one package.
With this integrated toolkit, researchers can streamline their workflow—from purification and IP to tag removal—saving valuable time while ensuring high-quality results.
Related Product
|
Category |
Name |
Cat.NO. |
Size |
|
Protein purification gel |
Anti-DYKDDDDK (Flag) Affinity Gel (Soluble or Membrane Protein) |
20585ES03/08/25/50 |
25 mL/100 mL |
|
20584ES03/08/25/60 |
1 mL/5 mL/ 25 mL/100 mL |
||
|
Eluted peptide |
Flag-tag Peptide |
20572ES08 |
5 mg |
|
Tag cleavage |
20395ES60/76/90 |
100 U/500 U/ 5000 U |
|
|
Protein purification magnetic beads |
Anti-DYKDDDDK (Flag) MagAgarose Beads |
20785ES03/08 |
1 mL/5 mL |
|
Immunomagnetic beads |
20565ES76/03/08 |
500 μL/1 mL/ 5 mL |
|
|
Immunoprecipitation (IP) Kit |
Anti-DYKDDDDK (Flag) IP/Co-IP Kit |
20765ES40 |
40T |