Endotoxin Removal Beads
Endotoxin Removal Beads are used to remove endotoxin in biological protein products (including polypeptides, antibodies and polysaccharides). The modified polymyxin B are attached to 4% agarose for specific removal of endotoxin. The endotoxin in the sample is reduced to 0.1 EU/ml, with a high protein recovery rate.
Table 1. Characteristics of Endotoxin Removal Beads
|
Item |
Description |
|
Matrix |
4% agarose |
|
Ligand |
Modified polymyxin B |
|
Binding capacity |
>2,000,000 EU/ml medium |
|
Particle size (μm) |
45-165 |
|
Maximum pressure |
0.1 MPa, 1 bar |
|
pH stability |
5-10 |
|
Chemical compatibilities |
20% DMSO,20% ethanol,20% glycerinum,1M urea, 300mM imidazole,0.05% Tween 20,10mM DTT |
|
Storage solution |
20% ethanol |
|
Storage |
2°C - 8°C |
2.1 Buffer Preparation
Please use endotoxin-free water and consumables to prevent the introduction of endotoxin during sample purification.
Binding Buffer: 20 mM phosphate, 0.15 M NaCl, pH 7.4
Regeneration Buffer: Binding Buffer containing 1% Triton X-114
Note: The Binding Buffer can be changed according to the properties of the samples. It is suggested that pH 7-8 and NaCl is about 0.15-0.5 M.
2.2 Sample Preparation
It is recommended to filter the sample solution by passing them through a 0.22 μm or 0.45 μm filter before use to reduce impurities, improve protein purification efficiency and prevent clogging of the column.
The pH of the sample is best controlled between pH 7-8, because the optimum pH for endotoxin binding to the beads is 6-9.
It is better to control the appropriate ion strength of the sample to reduce non-specific adsorption, such as 0.15-0.5 M NaCl.
2.3 Endotoxin removal
1) Mix the slurry by gently inverting the bottle several times to completely suspend the Endotoxin Removal Beads. Close the column outlet leaving the net covered with packing buffer. Transfer the slurry (1ml resin as an example) to the column using endotoxin-free tips.
2) Allow the resin to settle down and the buffer to drain from the column. Add 3 ml Regeneration Buffer onto the column to wash the beads. Control flow rate at 0.25 ml/min, or less than 10 drops per minute, and temperature at 2-8℃. Repeat at least twice to make sure there is no endotoxin in the column.
3) Add 3 ml Binding Buffer onto the column to equilibrate the beads. Control the flow rate at 0.5ml/min, the temperature at 2-8℃, and repeat at least twice.
4) Apply the pre-treated sample to the column and collect the flow-through after initial 1ml. Control flow rate at 0.25ml/min, or less than 10 drops per minute. Add 1ml Binding Buffer onto the column after entire sample enter the resin.
5) If the endotoxin in the sample is still above the target, repeat steps 1-4.
|
Problem |
Probable cause |
Solution |
|
Endotoxin removal is inefficient |
The pH value of the sample is not within the endotoxin binding range |
Adjust pH to 7-8 with 0.1MNaOH or 0.1M HCl.
|
|
Short contact time between sample and resin |
Reduce flow rate and increase sample contact time. |
|
|
Remove or detect system contaminated with endotoxin |
All System should be endotoxin-free.
|
|
|
Endotoxin strongly binds to target protein |
The pH of the sample was optimized to separate the sample from endotoxin. Increase contact time. |
|
|
Contaminated sample |
Resin was used for other samples |
Do not reuse resins to remove endotoxin for different samples. |
|
Low sample recovery |
The sample was nonspecific adsorbed on the resin |
Increase the concentration of NaCl in the sample and equilibrium solution.
|
|
The target protein is removed in combination with the endotoxin |
The pH of the sample was optimized to separate the sample from endotoxin. |
4. Related Products
|
Product |
Cat. No. |
Size |
|
Endotoxin Removal Beads |
SA031005 SA031025 SA031100 SA031500 SA03101L |
5 ml 25 ml 100 ml 500 ml 1 L |
