This product is an affinity purification medium created by coupling recombinant Peptostreptococcus magnus Protein L (rPPL) to a highly cross-linked agarose gel. Protein L from Peptostreptococcus magnus (PPL) binds specifically to the kappa light chain of antibodies without affecting the antigen-binding site. This property allows it to bind a broader range of antibodies from various sources and subclasses—such as human IgG, IgM, IgA, IgE, and IgD, as well as antibody fragments containing kappa light chains (human types 1, 3, 4; mouse type 1), including Fab and scFv—compared to Protein A and Protein G, which target the Fc region. However, it does not bind heavy chains, lambda light chains, or kappa light chains (type 2), and therefore cannot purify antibodies from bovine, goat, sheep, or chicken sources.
Aladdin rProtein L 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
| Recombinant Protein L, ≥6 mg/mL
|
Particle Size Range ①
| 45~165 μm
|
Average Particle Size
| ~90 μm
|
Dynamic Binding Capacity (DBC) ②
| ≥40 mg κ light chain h-IgG/mL resin
|
Recommended Operating Flow Rate ③
| 60~300 cm/h
|
Maximum Flow Rate & Pressure ④
| 900 cm/h，0.3 MPa
|
Operating pH Range
| 2–9 (recommended), 15 mM NaOH (CIP)
|
Chemical Stability
| Stable in: common aqueous buffers; 6 M guanidine HCl; 70% ethanol
|
Storage Conditions
| 20% ethanol, 2–8°C
|
Shelf Life
| 3 years
|
Notes:
① Over 90% of the beads fall within this size range.
② DBC10% tested under: 10% breakthrough, 6 min residence time.
③ This flow rate satisfies most usage scenarios with 2–6 min residence time across various column heights; not an exclusive operating range.
④ 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) Bring all materials to the operating temperature. Degas liquids if possible.
(2) Resin Quantity Calculation: Determine the settled resin volume required.
Settled resin volume = Column volume × Compression factor (1.15 for rProtein L 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. Alternatively, 20% ethanol + 0.4 M NaCl can be used as the packing solution.
(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 introducing air 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 recommended packing flow rates below; ensure pressure ≤0.3 MPa) until the interface is stable and clear. Mark the stable interface height. Stop the pump, open the top valve/fitting, close the bottom valve/fitting, and lower the adapter to the position corresponding to the compression factor (below the mark). Tighten the adapter. After packing, equilibrate the column at a high flow rate.
Packing Condition
| rProtein L Agarose Resin
|
Compression Factor
| 1.15
|
Packing Flow Rate
| 300–600 cm/h |
2. Column Efficiency Testing and Evaluation
After packing, column efficiency can be assessed using HETP (Height Equivalent to a Theoretical Plate) and As (Asymmetry Factor). Use acetone or NaCl as a test tracer under the following conditions:
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 the formulas:
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
Generally, HETP should be less than three times the average particle diameter (HETP/D₅₀ < 3), and As should be between 0.8–1.5.
3. Equilibration and Loading
A common buffer is PB Buffer (20 mM PB + 150 mM NaCl, pH 7.0–7.4), denoted as Buffer A.
Equilibrate the column with 5–10 CV of Buffer A at operating flow rate until baseline (conductivity/pH) stabilizes.
Load sample at 50–80% of DBC₁₀% (e.g., 20–32 mg h-IgG/mL resin for a DBC₁₀% of ~40 mg/mL). Centrifuge (≥10,000 g) and filter (0.22/0.45 μm) sample prior to loading.
After loading, wash with 3–10 CV of Buffer A.
4. Elution and Regeneration
Elute with a 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 antibody denaturation. Regenerate with 5–10 CV of elution buffer.
5. Cleaning-in-Place (CIP)
CIP with ≥2 CV of 15 mM NaOH (10–30 min contact time), followed by equilibration with ≥5 CV of Buffer A.
For stubborn contaminants (precipitated/hydrophobic proteins, lipids), use 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.
