This product is an affinity separation medium formed by covalently coupling a recombinant Protein A/Protein G fusion protein to agarose gel microspheres. Compared to media based solely on Protein A or Protein G, it exhibits a broader binding range and can bind all human IgG subclasses. It does not bind mouse IgA, IgM, or serum albumin, making it suitable for the extraction and detection of mouse IgG monoclonal antibodies. Furthermore, the fused rPAG protein shows reduced pH dependence, allowing binding within a pH range of 5–8.
Aladdin rProtein A/G Agarose Resin is stored in 20% ethanol, 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% cross-linked agarose
|
Ligand
| Protein A/G Fusion Protein, ≥6 mg/mL
|
Particle Size Range ①
| 45~165 μm
|
Average Particle Size
| ~90 μm
|
Binding Capacity ②
| ≥40 mg h-IgG/mL
|
Recommended Operating Flow Rate
| 60~150 cm/h
|
Maximum Flow Rate & Pressure ③
| 900 cm/h，0.3 MPa
|
Operating pH Range
| pH 3–9 (long-term), pH 2–10 (short-term)
|
Storage Conditions
| 20% ethanol, 2–8°C
|
Shelf Life
| 3年 |
Notes:
① Over 90% of the beads fall within this size range.
② Binding capacity tested under 2-hour incubation conditions.
③ Maximum tested flow rate at 10 cm column height.
Protocol
1. Column Packing
The following describes the packing procedure when connected to a chromatography system.
(1) Equilibrate all materials to the operating temperature. Degas liquids if possible.
(2) Resin Quantity Calculation:
Settled resin volume = Column volume × Compression factor (1.15 for this resin).
(3) Resin Preparation: Resuspend the resin slurry thoroughly. Measure the required volume, remove storage solution by filtration, and wash 3 times with ~3 resin volumes of purified water.
(4) Packing Slurry Preparation: Transfer resin to a suitable container and add packing solution to achieve a 50–75% (v/v) slurry. Mix well before use.
(5) Pre-packing Setup: Fill the bottom of the clean column with packing solution to remove air from the bottom frit and outlet. Retain a small amount of liquid, tighten the bottom end fitting, and ensure the column is vertical.
(6) Packing: Pour the well-mixed slurry into the column in one slow, continuous motion (use a packing reservoir if needed), allowing it to flow down the inner wall to avoid bubbles. After adding all resin, fill the reservoir with packing solution, attach the top lid, and connect the column to the system.
(7) Compression: Allow the resin to settle naturally (or use a low flow rate of 50–150 cm/h). Once settled (clear resin-liquid interface), remove the reservoir, attach the top adapter, and lower it to the interface. Apply a high flow rate (see table below; ensure pressure ≤0.3 MPa) until the interface is stable. Mark the stable height. Stop the pump, open the top valve, close the bottom valve, and lower the adapter to the position corresponding to the compression factor (below the mark). Tighten the adapter. Equilibrate the column at a high flow rate after packing.
Packing Condition
| rProtein A/G Agarose Resin
|
Compression Factor
| 1.15
|
Packing Flow Rate
| 300~600 cm/h |
2. Column Efficiency Testing and Evaluation
After packing, test column efficiency using HETP (Height Equivalent to a Theoretical Plate) and As (Asymmetry Factor). Use acetone or NaCl as a test tracer:
Tracer
| 1.0% Acetone
| 0.8~1.0 M NaCl
|
Sample Volume
| 1.0% CV
| 1.0% CV
|
Mobile Phase
| Pure Water
| 0.4 M NaCl
|
Flow Rate
| 30 cm/h
| 30 cm/h
|
Detector
| UV-280 nm
| Conductivity |
Calculate HETP, N (Number of Theoretical Plates), and As using:
HETP = L / N
N = 5.54 × (Vʀ / Wₕ)²
As = a / b
Where:
L = Column height
Vʀ = Retention volume
Wₕ = Peak width at half height
a = First half-width at 10% peak height
b = Second half-width at 10% peak height
HETP should be <3× the average particle diameter (HETP/D₅₀ < 3), and As should be 0.8–1.5.
3. Equilibration and Loading
Use PB Buffer (20 mM PB + 150 mM NaCl, pH 7.0–7.4) as Buffer A.
Equilibrate with 5–10 CV of Buffer A until baseline (conductivity/pH) stabilizes.
Load sample at 50–80% of DBC₁₀%. Centrifuge (≥10,000 g) and filter (0.22/0.45 μm) sample first.
Wash with 3–10 CV of Buffer A after loading.
4. Elution and Regeneration
Elute with low-pH buffer (e.g., glycine, citrate, or acetate, pH 2.5–3.0). Collect eluate into a neutralization buffer (e.g., 1 M Tris-HCl, pH 9.0) to minimize denaturation.
Regenerate with 5–10 CV of elution buffer to ensure complete removal of bound antibodies.
5. Cleaning-in-Place (CIP)
For contaminants (precipitated/hydrophobic proteins, lipids), wash with 2 CV of 0.1% Triton X-100 or 70% ethanol, followed by 5–10 CV of binding buffer.
6. Storage
Store in 20% ethanol at 2–8°C. Do not freeze.
ZJ25F1230739