The Flag tag is an octapeptide composed of hydrophilic amino acids, strategically positioned on the surface of fusion proteins. This location facilitates easier binding to antibodies and cleavage by enterokinase. Anti-Flag Agarose Resin utilizes an anti-Flag (DYKDDDDK) antibody as the affinity ligand for the one-step purification of Flag-tagged fusion proteins expressed in prokaryotic, yeast, or mammalian cell systems. This product is based on a 4% agarose gel matrix, which minimizes non-specific binding of host cell proteins, making it suitable for both the purification and immunoprecipitation (IP) of Flag-tagged fusion proteins.
Aladdin Anti-Flag Agarose Resin is stored in a solution containing 0.1% ProClin 300, with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel.
Parameter
| Value / Description
|
Matrix
| 4% Agarose Microspheres
|
Ligand
| Anti-DYKDDDDK Antibody
|
Particle Size Range
| 45~165 μm |
| Binding Capacity | >1 mg DYKDDDDK-tagged protein / mL resin
|
Maximum Pressure
| 0.1 MPa, 1 bar |
Storage Conditions
| 0.1% ProClin 300, 2~8℃
|
Shelf Life
| 2 years
|
Protocol
1. Sample Preparation
Ensure the sample solution has appropriate ionic strength and pH before loading. Dilute the sample or cell culture supernatant with equilibration buffer, or dialyze the sample against equilibration buffer.
Clarify the sample by centrifugation or filtration through a 0.22 μm or 0.45 μm membrane to reduce impurities, improve purification efficiency, and prevent column clogging.
2. Buffer Preparation
It is recommended to filter water and buffers through a 0.22 μm or 0.45 μm membrane before use.
Equilibration/Wash Buffer: 50 mM Tris, 0.15 M NaCl, pH 7.4
Acidic Elution Buffer: 0.1 M Glycine-HCl, pH 3.0
Competitive Elution Buffer: 50 mM Tris, 0.15 M NaCl, 100-500 μg Flag peptide / mL, pH 7.4
Neutralization Buffer: 1 M Tris-HCl, pH 8.0
3. Sample Purification
3.1 Column Chromatography
(1) Pack the Anti-Flag Agarose Resin into a suitable chromatography column. Equilibrate the column with 5 column volumes (CV) of Equilibration Buffer to bring the resin into the same buffer system as the target protein.
(2) Load the sample onto the equilibrated Anti-Flag Agarose Resin. Collect the flow-through. The sample can be reloaded to increase binding efficiency.
(3) Wash with 10 CV of Wash Buffer to remove non-specifically bound proteins. Collect the wash fractions.
(4) Elution:
* A. Acidic Elution: Elute with 5 CV of Acidic Elution Buffer. Add a volume of Neutralization Buffer equal to one-tenth of the elution volume to each fraction to adjust the pH to 7.0–8.0. Collect fractions separately.
* Note: After acidic elution, the resin must be immediately re-equilibrated. Do not expose the Anti-Flag Agarose Resin to the acidic elution buffer for more than 20 minutes.**
* B. Competitive Elution: Elute with 5 CV of Competitive Elution Buffer. Collect fractions separately.
(5) Regenerate the resin with 3 CV of the respective Elution Buffer, then re-equilibrate with Equilibration Buffer until neutral pH is reached.
(6) Store the resin in Storage Buffer at 2–8°C.
3.2 Batch/Binding Method
(1) Resin Preparation: Transfer an appropriate amount of Anti-Flag Agarose Resin to a column and drain the storage solution. Wash with 5 CV of Equilibration Buffer.
(2) Add the sample solution. Incubate with shaking at 4°C or room temperature for at least 30 minutes (avoid magnetic stirring). Ensure thorough mixing of the resin and sample.
(3) After incubation, centrifuge the mixture (5,000 × g, 1 min) or filter to collect the resin.
(4) Transfer the resin to a column. Wash with Equilibration Buffer until the UV baseline stabilizes.
(5) Elute using either the Acidic or Competitive Elution method as described in section 3.1 (4).
(6) Regenerate and store the resin as described in sections 3.1 (5) and (6).
3.3 Immunoprecipitation (IP) Procedure
(1) Resin Preparation: Add 40 µL of Anti-Flag Agarose Resin suspension (20 µL settled resin) to a 2 mL tube. Centrifuge at 5,000 × g for 1 min. Carefully remove and discard the supernatant.
(2) Add 0.5 mL of Equilibration Buffer to resuspend the resin (this brings it into the correct buffer system, protecting the protein). Centrifuge at 5,000 × g for 1 min. Discard the supernatant. Repeat this wash step once.
(3) Add 200–1000 µL of sample lysate to the prepared resin. Mix thoroughly and incubate on a tube rotator or roller mixer at room temperature for at least 1 hour to facilitate binding. Centrifuge at 5,000 × g for 1 min. Discard the supernatant.
(4) Washing: Add 0.5 mL of Wash Buffer, resuspend the resin, and mix gently. Centrifuge at 5,000 × g for 1 min. Discard the supernatant. Repeat this wash step three more times to ensure removal of non-specifically bound material.
(5) Elution: Choose the elution method based on downstream application requirements.
* A. Acidic Elution: Add 100 µL of Acidic Elution Buffer and resuspend the resin. Incubate at room temperature for 5 min. Centrifuge at 5,000 × g for 1 min. Carefully collect the supernatant without disturbing the resin. Neutralize immediately with Neutralization Buffer. Store eluted samples at 4°C short-term or -20°C long-term.
* B. Competitive Elution: Add 100 µL of Competitive Elution Buffer and resuspend the resin. Incubate at room temperature for 30 min. Centrifuge at 5,000 × g for 1 min. Carefully collect the supernatant. Store eluted samples at 4°C short-term or -20°C long-term.
* C. Denaturing Elution (SDS-PAGE): Standard protein loading buffer (containing β-mercaptoethanol/DTT and SDS) will denature the anti-Flag antibody, releasing the bound protein but rendering the resin unusable for reuse. Add 20 µL of 2× Loading Buffer to the resin, heat at 95°C for 5 min. Centrifuge at 5,000 × g for 1 min, and load the supernatant directly onto an SDS-PAGE gel for analysis.
Reagent Compatibility
Reagent
| Maximum Tolerant Concentration
| Notes
|
β-Mercaptoethanol
| 10 mM
| Avoid during purification; if used in IP, resin cannot be reused
|
DTT
| 80 mM
| Avoid during purification; if used in IP, resin cannot be reused
|
SDS
| --
| Avoid during purification; if used in IP, resin cannot be reused
|
EDTA
| 5 mM
| Higher concentrations reduce protein recovery
|
Tween-20
| 5%
| High concentrations may reduce binding efficiency
|
Triton X-100
| 5%
| High concentrations may reduce binding efficiency
|
NP-40
| 4%
| High concentrations may reduce binding efficiency
|
Guanidine HCl
| 0.3 M
| Higher concentrations denature the antibody
|
Urea
| 1.5 M
| Higher concentrations denature the antibody
|
Glycerol
| 20%
| High concentrations may affect protein binding
|
NaCl
| 1 M
| Helps reduce non-specific adsorption |
Troubleshooting Guide
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