His-tag Purification Resins (BG IDA FF series)

Operation Methods

The chromatographic operation of His-tag Purification Resins(IDA series) usually includes five steps such as equilibration, loading, washing, elution, and regeneration. The specific operations for each step are as follows:

1. Equilibration: Equilibrate the chromatography column with 5～10CV of equilibration buffer until the conductivity and pH of the effluent remain constant (consistent with the equilibration buffer). In practical operation, neutral/weakly alkaline (pH 7～8) high-salt (0.15～1.0 M NaCl or other neutral salt) buffer is commonly used. Phosphate buffer system is commonly chosen( 20 mM PB+0.5 M NaCl, pH 7.4.) For proteins with strong binding histidine tags, low concentration (20～40 mM) imidazole can be added to the equilibration buffer.

2. Sample loading: The conductivity and pH of loading sample should be as consistent as possible with the equilibration buffer. Solid samples can be dissolved and prepared in equilibration buffer; low-concentration sample solutions can be dialyzed with equilibration buffer; high-concentration sample solutions can be diluted with equilibration buffer. To avoid blocking the column, samples should be centrifuged or microfiltered. The loading volume can be calculated based on the binding capacity and the target protein content. To reduce non-specific protein adsorption on the column, imidazole concentration in the sample buffer can be appropriately increased while ensuring target protein adsorption. For inclusion body proteins, 8M urea or 6M guanidine hydrochloride can be added to the buffers for equilibration, sample loading, and elution accordingly.

3. Washing: After sample loading, column should be continually washed with equilibration buffer until the UV absorbance drops to baseline.

4. Elution: There are generally two methods of elution: competitive reagents elution and pH elution. Imidazole (0-0.5M), histidine (0-0.05M), or ammonium chloride (0-2M) can be used as competitive reagents to displace proteins from the column; target proteins can also be eluted by adjusting the pH value, as most proteins can be eluted at pH 4-6. When using competitive reagents imidazole or pH reduction to elute proteins, metal ions remain bound to the column; if histidine or ammonium chloride is used, both metal ions and protein complexes will be eluted together. The elution buffer should contain 0.15-1.0 M NaCl to eliminate ion exchange effects. Gradient elution should be performed at a constant pH of the initial equilibration buffer, using either linear or step gradients.

5. Regeneration: After multiple uses (depending on characteristics of loading samples), or before chelating other metal ions, regeneration is required.

Ni removal: Clean the medium with 5 volumes of EDTA solution (0.2M EDTA + 0.5M NaCl, pH 7.0) at a low flow rate (recommended at half the normal loading rate), followed by 5 volumes of 0.5M NaCl to remove residual EDTA.

Cleaning: Use 2-3 volumes of 2M NaCl to remove proteins bound by ionic bonds, followed by 3 volumes of pure water. The column can be cleaned with 5 volumes of 0.5-1M NaOH (or 2M NaCl solution) to remove pigments or strongly adsorbed proteins, then equilibrate with 5-10 volumes of 0.2M PBS buffer (monitor pH if possible, until pH reaches buffer value).Clean with 5-10 volumes of 70% ethanol or 30% isopropanol (for at least 20 minutes), followed by 3-10 volumes of pure water. Note that isopropanol may require larger volumes for thorough removal. Alternatively, 2 volumes of detergent-containing alkaline or acidic solution can be used, for example: wash with 0.1～0.5% non-ionic detergent + 0.1M acetic acid for 1～2 hours, followed by washing with 5～10 volumes of 70% ethanol to remove the detergent, and finally wash with 3～10 volumes of pure water.

Ni loading: Mix 3-5 volumes of 0.1M NiSO4 solution with the resins and stir for 4 hours, then filter. (Alternatively, pass 3-5 column volumes of 0.1M NiSO4 solution through the packed column at a low flow rate, cycling the NiSO4 solution three times to ensure complete loading.)

Equilibration: Clean the column with 3 volumes of deionized water, then rinse with 5-10 volumes of 0.25M imidazole + 0.5M NaCl solution to remove residual Ni2+, finally clean with 5 volumes of deionized water.

6. Storage: Clean the column with 2-5 volumes of 20% ethanol and store at 2-30°C.

7. Other considerations: Avoid column dry-out or leaks during use and storage to prevent air bubbles. 

3.Specification