Do any substances interfere with the binding of LPS to Detoxi-Gel Support?
Yes. Other detergents and high levels of chaotropes (urea and guanidine) can reduce the affinity of the Polymixin B for the LPS. Proteins such as BSA can bind tightly to endotoxin, reducing its ability to interact with and bind to the Detoxi-Gel Support. This reduction in binding capacity can sometimes be overcome by increasing the volume of Detoxi-Gel Support to LPS. However, some proteins bind tightly to endotoxin without inhibiting its binding to the support. In this case, the protein will remain bound to the gel with the endotoxin, possibly causing a loss of the protein of interest.
How many times can the same Detoxi-Gel Endotoxin Removing Gel be used?
The columns may be reused at least 10 times without loss of activity.
Is it possible for substances other than LPS to bind to the Detoxi-Gel Support?
Yes. Hepatitis B surface antigen, for example, is known to stick to the column. Adding 0.5 M NaCl to the sample and elution buffers can sometimes break the affinity for certain substances that bind nonspecifically, releasing them from the gel.
What is Detoxi-Gel Endotoxin Removing Gel, and how does it work?
Detoxi-Gel Support consists of 6% cross-linked beaded agarose that has Polymixin B immobilized onto it. Polymixin B is an antibiotic that contains a cationic cyclopeptide with a fatty acid chain that can neutralize the biological activity of endotoxins by binding to the lipid A portion of the bacterial lipopolysacharide (LPS). After the endotoxin from a sample binds to the immobilized Polymixin B, the sample can be collected and the endotoxin can be eluted using 1% sodium deoxycholate.
What is the binding capacity of Detoxi-Gel Endotoxin Removing Gel?
Detoxi-Gel Support has the capacity to bind up to 2µg LPS/ml of gel. Because one endotoxin unit (EU) is approximately 0.1 ng/ml, the gel has the potential for binding up to 20,000 EU/ml of gel.
What is the protocol for using Detoxi-Gel Endotoxin Removing Gel?
- Equilibrate the column with 5 column volumes of 1% sodium deoxycholate in pyrogen-free water, followed by 3 column volumes of pyrogen-free water or buffer of choice.
- Apply sample, being sure not to exceed the binding capacity of the gel.
- Collect sample by gravity flow by applying additional pyrogen-free water or buffer.
- Regenerate the column with 5 column volumes of 1% sodium deoxycholate in pyrogen-free water, followed by 3 column volumes of pyrogen-free water.
What is the void volume of the column?
The Pierce 1 ml Detoxi-Gel Column has a void volume of 0.3-0.5 ml. This is the volume of solution that must be collected from the column before the sample will begin to come off the column.
Why must I regenerate the column before and after each use?
It is possible that the column may have been contaminated with endotoxin during storage and handling. Equilibrating the column with 1% sodium deoxycholate followed by pyrogen-free washing ensures that the column is free of endotoxin before the sample is added. The column must be regenerated following endotoxin removal from a sample to make sure the endotoxin that has bound to the column from the sample is washed off.