The HA tag consists of the amino acid sequence YPYDVPDYA (residues 98-106 of human influenza hemagglutinin). It has minimal impact on the tertiary structure of the target foreign protein and can be easily fused to either the N- or C-terminus, making it a popular choice for recombinant protein expression. Anti-HA Agarose Resin 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 HA-tagged fusion proteins.
Aladdin Anti-HA 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-HA Mouse Monoclonal Antibody
|
Particle Size Range
| 45~165 μm |
| Binding Capacity | >1 mg HA-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 Buffer: 10 mM Tris, 0.15 M NaCl, pH 7.4
Wash Buffer: 10 mM Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7.4
Chemical Elution Buffers:
Competitive Elution Buffer: 50 mM Tris, 0.15 M NaCl, 100–500 µg HA peptide / mL, pH 7.4
Neutralization Buffer: 1 M Tris-HCl, pH 8.5
Comparison of Chemical Elution Buffers
| Solution | Advantages | Disadvantages
|
0.1M glycine HCl, pH 2.0-2.8
| Does not damage resin binding capacity if target protein is stable at low pH
| Low elution efficiency; target protein may denature
|
3M NaSCN
| High elution efficiency; does not damage resin binding capacity
| Target protein may denature
|
50mM NaOH
| High elution efficiency
| Target protein may denature; reduces resin lifespan
|
3. Sample Purification
3.1 Column Chromatography
(1) Pack the Anti-HA Agarose Resin into a suitable chromatography column. Equilibrate the column with 5 column volumes (CV) of Equilibration Buffer.
(2) Load the sample onto the equilibrated resin. Collect the flow-through. The sample can be reloaded to increase binding efficiency.
(3) Wash with 10–20 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 (e.g., 0.1 M Glycine-HCl, pH 2.0–2.8). 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 resin to the acidic elution buffer for more than 20 minutes.
* B. Chemical Elution: Elute with 5 CV of a chemical elution buffer (e.g., 3 M NaSCN or 50 mM NaOH). Collect fractions separately.
* Note: After chemical elution, the resin must be immediately re-equilibrated. Do not expose the resin to the chemical elution buffer for more than 20 minutes.
* C. Competitive Elution: Elute with 5 CV of Competitive Elution Buffer. Collect fractions separately.
(5) Regenerate the resin with 3 CV of a chemical elution buffer (e.g., Glycine-HCl), 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-HA 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 30 minutes (avoid magnetic stirring). Ensure thorough mixing.
(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 Chemical 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-HA Agarose Resin suspension (20 µL settled resin) to a 1.5 mL tube. Centrifuge at 5,000 × g for 1 min. Discard the supernatant.
(2) Add 0.5 mL of Equilibration Buffer to resuspend the resin. 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 and incubate on a tube rotator at room temperature for at least 1 hour. Centrifuge at 5,000 × g for 1 min. Collect the supernatant for analysis.
(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.
(5) Elution: Choose the elution method based on downstream application.
* A. Chemical Elution: Add 100 µL of chemical elution buffer (0.1 M Glycine-HCl pH 2.0-2.8, 3 M NaSCN, or 50 mM NaOH) and resuspend the resin. Incubate at room temperature for 5 min. Centrifuge at 5,000 × g for 1 min. Carefully collect the supernatant and neutralize immediately if acidic. 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. Repeat elution 1-2 times. Store eluted samples at 4°C short-term or -20°C long-term.
* C. Denaturing Elution (SDS-PAGE): Add 20 µL of 2× Loading Buffer (contains SDS and reducing agents like β-mercaptoethanol/DTT) 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. Note: This method denatures the antibody, rendering the resin unusable for reuse.
Troubleshooting Guide
