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Epitope-tagged Protein Purification
We offer multiple supports for the efficient purification of DYKDDDDK (FLAG)-, and c-Myc-, HA-tagged proteins using immobilized anti-tag antibodies. Our portfolio is designed to meet small-scale (screening) to pilot scale needs.
- Specific—highly specific monoclonal antibody-based ligands enable high yield and high purity for immunoprecipitation or small to pilot scale protein purification.
- Low non-specific binding—stable beads and target-specific antibody minimize off-target binding
- Scalable—available in larger package sizes for purification scale-up and more economical pricing
- Versatile—multiple resin formats available that can be used in spin or gravity columns as well as in FPLC cartridges
- Automation friendly—magnetic agarose or beads recommended for high throughput applications
- Convenient—reagents to elute and detect tagged fusion proteins are available separately
Choose a product for epitope tag immunoaffinity purification
| Pierce Anti-DYKD4K (FLAG) Magnetic Agarose | Pierce Anti-DYKD4K (FLAG) Affinity Resin UltraLink | Pierce Anti-c-Myc Magnetic Beads | Pierce Anti-c-Myc Agarose (Superflow 6) | Pierce Anti-HA Magnetic Beads | Pierce Anti-HA Agarose | |
|---|---|---|---|---|---|---|
| Bead or resin size | 10 - 40 uM | 50 - 80 um | 1 uM | 45-165 μm | 1 uM | 45-165 μm |
| Binding Capacity | ≥3 mg/mL | ≥ 3 mg/mL | ≥ 100 μg/mL | 60-150 nmol/mL | ≥ 100 μg/mL | 102 - 144 nmol/mL |
| Maximum Linear Flow rates (cm/hr) | N/A | 3,000 | N/A | 1,200 | N/A | 700 |
| Support | Magnetite-embedded 6% beaded agarose | Durable beaded polyacrylamide support | Magnetite-coated polymer | Highly crosslinked 6% beaded agarose | Magnetite-coated polymer | 4% beaded agarose |
| Recommended Application scale | High throughput batch | Batch, pilot | High throughput screening | Batch, pilot | High throughput screening | Batch |
| Formats available | loose bead | loose resin | loose bead | loose resin | loose bead | loose resin |
| Recommended application scale | screen | batch, pilot | batch, pilot | screening | batch, pilot | batch, pilot |
| A36797 | A36801 | 88842 | 20168 | 88836 | 26181 |
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Figure 1. Comparison of DYKDDDDK-tagged SUMO protein purification results using Pierce Anti-DYKDDDDK supports and other suppliers. C- and N-terminal DYKDDDDK-tagged SUMO protein were expressed in E. coli and purified using two Pierce Anti-DYKDDDDK and other suppliers’ supports, Tagged protein was competitively eluted with 3x DYKDDDDK peptide and analyzed by SDS-PAGE (A, C) and densitometry (B, D) using the Invitrogen iBright Imaging system. The results for the Pierce Anti-DYKDDDDK Magnetic Agarose (A, B) and Pierce Anti-DYKDDDDK Affinity Resin (C,D) and other suppliers are shown. The comparison of the starting lysate and elution fractions show effective capture and elution of DYKDDDDK-tagged protein with minimal background by the Pierce supports compared to other suppliers.
Click to enlarge
Figure 2. Comparison of protein purification results between Pierce Anti-DYKDDDDK magnetic agarose and another supplier. C-terminal DYKDDDDK-tagged Green Renilla Luciferase protein was expressed using the Thermo Scientific 1-Step Human High-Yield Maxi IVT Kit and immunoprecipitated using Pierce Anti-DYKDDDDK Magnetic Agarose or Sigma-Aldrich Anti-FLAG™ M2 Magnetic Beads using the KingFisher Flex Purification System. Tagged proteins were competitively eluted with Pierce 3x DYKDDDDK peptide and analyzed by western blot (A), and silver staining (C) and Pierce Renilla Luciferase Glow Assay (B). Comparison of the starting lysate (L), elutions (E), and bead boiled samples (BB) show effective capture and elution of DYKDDDDK-tagged proteins with no background. Correlation of protein and activity levels indicate that a high level of Green Renilla luciferase activity is maintained after purification and competitive peptide elution.
Figure 3. Dynamic binding capacity versus residence time. Pierce Anti-DYKDDDDK Affinity Resin and Sigma Anti-FLAG™ M2 Affinity Gel were packed into 1 mL columns (0.5 cmD x 5 cmL) and loaded with purified DYKDDDDK-TurboGFP-His (1 mg/mL) in 100mM phosphate, 150mM NaCl, pH 7.2 (PBS) under a variety of resident times (150 cm/hr, 60 cm/hr, and 30 cm/hr) until a 10% breakthrough was achieved as measured by A280.
Figure 4. Better immunoprecipitation results with Thermo Scientific Pierce Anti-HA Magnetic Beads. Using a KingFisher Flex Instrument with 96 deep-well plates, 25 μL of Pierce Anti-HA Magnetic Beads, AntiHA-tag Magnetic Beads (MBL International Corp.), and SPHERO™ Rabbit Anti-HA Magnetic Beads (Spherotech Inc.) were used to immunoprecipitate GST-PI3K-SH2-HA from 50 μg of E. coli lysate in duplicate. Captured protein was eluted with 0.1 M glycine, pH 2.0, and then resolved by SDSPAGE and analyzed by western blot for the HA-tagged protein.
Figure 5. Better immunoprecipitation results with Thermo Scientific Pierce Anti-c-Myc Magnetic Beads. Green Renilla luciferase c-Myc fusion protein was expressed in 293T cells. For IP, identical aliquots of the cell lysate were incubated in duplicate for one hour at room temperature with anti-c-Myc magnetic beads from each manufacturer. For all conditions, IP products were eluted identically using low-pH buffer. Eluted fractions (25 µL each) were separated by 12% SDS-PAGE, transferred to PVDF membranes, and detected via anti-c-Myc antibody (Cat. No. MA1-980), goat anti-mouse secondary antibody, and chemiluminescent substrate (Cat. No. 34080).
Click to enlarge
Figure 1. Comparison of DYKDDDDK-tagged SUMO protein purification results using Pierce Anti-DYKDDDDK supports and other suppliers. C- and N-terminal DYKDDDDK-tagged SUMO protein were expressed in E. coli and purified using two Pierce Anti-DYKDDDDK and other suppliers’ supports, Tagged protein was competitively eluted with 3x DYKDDDDK peptide and analyzed by SDS-PAGE (A, C) and densitometry (B, D) using the Invitrogen iBright Imaging system. The results for the Pierce Anti-DYKDDDDK Magnetic Agarose (A, B) and Pierce Anti-DYKDDDDK Affinity Resin (C,D) and other suppliers are shown. The comparison of the starting lysate and elution fractions show effective capture and elution of DYKDDDDK-tagged protein with minimal background by the Pierce supports compared to other suppliers.
Click to enlarge
Figure 2. Comparison of protein purification results between Pierce Anti-DYKDDDDK magnetic agarose and another supplier. C-terminal DYKDDDDK-tagged Green Renilla Luciferase protein was expressed using the Thermo Scientific 1-Step Human High-Yield Maxi IVT Kit and immunoprecipitated using Pierce Anti-DYKDDDDK Magnetic Agarose or Sigma-Aldrich Anti-FLAG™ M2 Magnetic Beads using the KingFisher Flex Purification System. Tagged proteins were competitively eluted with Pierce 3x DYKDDDDK peptide and analyzed by western blot (A), and silver staining (C) and Pierce Renilla Luciferase Glow Assay (B). Comparison of the starting lysate (L), elutions (E), and bead boiled samples (BB) show effective capture and elution of DYKDDDDK-tagged proteins with no background. Correlation of protein and activity levels indicate that a high level of Green Renilla luciferase activity is maintained after purification and competitive peptide elution.
Figure 3. Dynamic binding capacity versus residence time. Pierce Anti-DYKDDDDK Affinity Resin and Sigma Anti-FLAG™ M2 Affinity Gel were packed into 1 mL columns (0.5 cmD x 5 cmL) and loaded with purified DYKDDDDK-TurboGFP-His (1 mg/mL) in 100mM phosphate, 150mM NaCl, pH 7.2 (PBS) under a variety of resident times (150 cm/hr, 60 cm/hr, and 30 cm/hr) until a 10% breakthrough was achieved as measured by A280.
Figure 4. Better immunoprecipitation results with Thermo Scientific Pierce Anti-HA Magnetic Beads. Using a KingFisher Flex Instrument with 96 deep-well plates, 25 μL of Pierce Anti-HA Magnetic Beads, AntiHA-tag Magnetic Beads (MBL International Corp.), and SPHERO™ Rabbit Anti-HA Magnetic Beads (Spherotech Inc.) were used to immunoprecipitate GST-PI3K-SH2-HA from 50 μg of E. coli lysate in duplicate. Captured protein was eluted with 0.1 M glycine, pH 2.0, and then resolved by SDSPAGE and analyzed by western blot for the HA-tagged protein.
Figure 5. Better immunoprecipitation results with Thermo Scientific Pierce Anti-c-Myc Magnetic Beads. Green Renilla luciferase c-Myc fusion protein was expressed in 293T cells. For IP, identical aliquots of the cell lysate were incubated in duplicate for one hour at room temperature with anti-c-Myc magnetic beads from each manufacturer. For all conditions, IP products were eluted identically using low-pH buffer. Eluted fractions (25 µL each) were separated by 12% SDS-PAGE, transferred to PVDF membranes, and detected via anti-c-Myc antibody (Cat. No. MA1-980), goat anti-mouse secondary antibody, and chemiluminescent substrate (Cat. No. 34080).
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