Product Introduction
Protein At Beads LX is an affinity chromatography medium for the separation and purification of monoclonal antibodies, polyclonal antibodies, or Fc-fusion proteins. Detailed performance is shown in Table 1.
Protein A is a cell wall protein isolated from Staphylococcus aureus, which mainly binds to mammalian IgG through the Fc fragment. The ligand protein of Protein At Beads LX is an alkali-tolerant Protein A (At) obtained by bioengineering mutation based on native Protein A and expressed in Escherichia coli. No animal-derived components are used during ligand purification. The specially designed ligand enhances stability against alkali and proteases.
Protein At Beads LX exhibits a high dynamic binding capacity after extended residence time, and is specially designed for the purification of high-titer antibodies. Features including alkali tolerance, high binding capacity, low ligand leakage, and a highly cross-linked 4% agarose gel rigid matrix make this medium particularly suitable for large-scale industrial or clinical antibody purification.
MabCap At LX is a medium-pressure pre-packed column available in 1 mL and 5 mL sizes, filled with 1 mL and 5 mL of Protein At Beads LX respectively. There are 5 package specifications in total. The pre-packed column has standard interfaces compatible with various commercial medium-pressure chromatography systems (e.g., ÄKTA), facilitating operation. Detailed performance is listed in Table 1.
Table 1. MabCap At LX Product Information
| Item |
Specification |
| 5 mL Column Size (ID × Height) |
1.6 × 2.5 cm |
| Matrix |
Highly cross-linked agarose beads |
| Ligand |
Protein A |
| Binding Capacity* |
> 60 mg Human IgG/mL medium |
| Particle Size |
45–165 μm |
| Pressure Resistance |
0.3 MPa, 3 bar |
| Chemical Stability |
Compatible with common reagents in antibody purification |
| Working pH |
3–12 |
| Cleaning-in-Place (CIP) |
0.1–0.5 M NaOH |
| Linear Flow Rate |
50–500 cm/h |
| Storage |
1×PBS, 2–8 °C |
*Note: The dynamic binding capacity for the target antibody must be determined using front-end analysis with actual samples. Dynamic binding capacity is a function of sample residence time and should be defined across different residence time ranges.
2. Purification Protocol
2.1 Buffer Preparation
All water and buffers are recommended to be filtered through 0.22 μm or 0.45 μm membranes before use.
- Equilibration/Wash Buffer: 0.15 M NaCl, 20 mM Na₂HPO₄, pH 7.0
- Elution Buffer: 0.1 M Glycine, pH 3.0–3.6
- Neutralization Buffer: 1 M Tris-HCl, pH 8.5
2.2 Sample Preparation
Ensure the sample solution has appropriate ionic strength and pH before loading. Dilute serum, ascites, or cell culture supernatant with equilibration/wash buffer, or dialyze the sample against equilibration/wash buffer.
Samples are recommended to be centrifuged or filtered (0.22/0.45 μm) before loading to reduce impurities, improve purification efficiency, and prevent column clogging.
2.3 Sample Purification
MabCap At LX is a pre-packed column for separating and purifying monoclonal/polyclonal antibodies or Fc-fusion proteins, compatible with standard medium/low-pressure chromatography systems.
- Fill pump tubing with deionized water. Remove the top plug, connect the column to the chromatography system, open the bottom outlet, and tighten the connection.
- Flush storage buffer with 3–5 column volumes (CV) of deionized water.
- Equilibrate the column with at least 5 CV of equilibration buffer.
- Load sample via pump or sample loop; ensure residence time > 6 minutes.
Note: Increased sample viscosity may cause high backpressure even with small loading volumes. Do not overload beyond the column binding capacity. Large sample volumes may also raise backpressure.
- Wash the column with wash buffer until UV absorbance reaches a stable baseline (typically 10–15 CV).
- Elution:
- Linear gradient elution: 0–100% elution buffer over 10 CV; collect eluted fractions containing target protein. Linear elution is recommended for first-time use to optimize elution pH and protect labile antibody activity.
- Isocratic elution: After initial method development, 5–10 CV of fixed-pH elution buffer can be used for scale-up. This concentrates eluted antibody, reduces buffer consumption, and shortens cycle time.
Immediately neutralize eluted fractions to neutral pH; typically add 1/10 volume of neutralization buffer.
Note: For first use, perform CIP cleaning (Section 4) to remove residual leached ligand.
- After elution: flush with 3 CV equilibration buffer → 5 CV deionized water → 2 CV 20% ethanol; store at 2–8 °C.
2.4 SDS-PAGE Analysis
Analyze purification performance by SDS-PAGE using raw sample, flow-through, wash, and eluted fractions.
3. Residual Ligand Removal
Protein At Beads LX shows very low ligand leakage (< 10 ng/mg antibody). For applications requiring complete ligand removal, use cation exchange, anion exchange, or gel filtration chromatography; refer to the instructions for corresponding media.
4. Medium Cleaning
Protein At Beads LX can be reused without regeneration. However, denatured precipitate and protein aggregation may reduce flow rate and binding capacity, increase backpressure, or cause ligand leakage, severely impairing column performance. In such cases, clean the medium to restore capacity, flow rate, and performance.
CIP Cleaning
Protein At Beads LX is alkali-tolerant and can be cleaned with 0.1–0.5 M NaOH (low cost, high efficiency):
- 3 CV equilibration buffer
- At least 2 CV 0.1–0.5 M NaOH; contact time 15 minutes
- 5 CV equilibration buffer
Note: High viscosity of 0.1–0.5 M NaOH may increase backpressure; reverse flushing is recommended.
5. Troubleshooting
| Problem |
Cause |
Recommended Solution |
| High column backpressure |
Sieve plate clogged |
Clean or replace sieve plate |
| Medium clogged |
Perform CIP cleaning (Section 4) |
| Lysate contains fine solid particles |
Filter (0.22/0.45 μm) or centrifuge sample before loading |
| Unstable UV profile during purification |
Air bubbles in sample/buffer |
Degas sample and buffers; remove air from column |
| No target protein in eluate |
Low antibody concentration in sample |
Use antigen-based affinity medium |
| Antibody degraded |
Increase elution pH appropriately |
| Gradually decreasing recovery |
Overloading |
Reduce sample loading amount |
6. Ordering Information & Related Products
| Product Name |
Cat. No. |
Size |
| Protein At Beads LX |
SA085005 |
5 mL |
| SA085025 |
25 mL |
| SA085100 |
100 mL |
| SA085500 |
500 mL |
| SA08501L |
1 L |
| SA08510L |
10 L |
| MabCap At LX |
SA085C11 |
1×1 mL |
| SA085C51 |
5×1 mL |
| SA085C15 |
1×5 mL |
| SA085C55 |
5×5 mL |
| SA085CS |
3×1 mL + 1×5 mL |
