How to effectively remove endotoxin? -Huaqiang Electronic Network

Endotoxin

Endotoxin

), also known as lipopolysaccharide or LPS, is a cell membrane component of Gram-negative bacteria such as E. coli. The lipid portion of the outer membrane outer layer is composed entirely of endotoxin molecules. A single E. coli cell contains approximately 2 million LPS molecules, each consisting of a hydrophobic lipid A, a sugar residue, and a negatively charged phosphate group complex. Thus, each endotoxin molecule has hydrophobic, hydrophilic, and charged regions that result in its unique interaction with other molecules. Bacteria secrete a small amount of endotoxin into the surrounding environment during active growth or massive death. When lysing bacterial cells for plasmid preparation, endotoxin molecules are released from the outer membrane into the lysate.

Effect of endotoxin on biological applications

Endotoxin strongly affects DNA transfection into primary cells and sensitive cultured cells, and increased levels of endotoxin result in a dramatic decrease in transfection efficiency. Furthermore, the use of endotoxin-free plasmid DNA for gene therapy is extremely important because endotoxin can cause activation of animal and human fever, endotoxic shock syndrome and the complement cascade. Endotoxin also interferes with in vitro immunization into immune cells such as macrophages and B cells by activating non-specific immune responses. These reactions include the induction synthesis of immune mediators such as IL-1 and prostaglandins. Ensure that plastic products, media, serum, and plasmid DNA are free of LPS contamination and avoid misleading results.

Endotoxin assay

Classical endotoxin assays are based on the clotting reaction between endotoxin and clotting proteins in tendon morphing cells. More sensitive photometric assays (e.g., Kinetic-QCL Test from BioWhittaker, Inc) can now be used based on sputum metamorphic cell lysate (LAL) and synthetic color producing substrates. LPS contamination is usually expressed in endotoxin units (EU). Pass, 1 ng LPS corresponds to 1-10 EU.

Endotoxin removal

Today, endotoxin removal uses commercial kits. Polymyxin B (polymixin B) is typically used to attach to agarose microspheres for specific endotoxin removal. Polymyxin B can adsorb endotoxin by various factors such as electrostatic action, ionic action, hydrophilic action and interaction between molecules, so as to achieve the purpose of endotoxin removal:

Product Name Product Specification Product No. Product Details PurKine Endotoxin Removal Kit (Polycide B)-PurKineEndotoxinRemovalKit(polymyxinB) 1ml×5/3ml×5 KTP21400 Details PurKine Endotoxin Removal Resin-PurKineEndotoxinRemovalResin 5ml/100ml BMR21400 Details PurKine Endotoxin Clear Prepacked Column - PurKineEndotoxinRemovalPackedColumn 1ml×5/3ml×5 BMC21400 Details

In the process of endotoxin removal, we must remember to use endotoxin-free plastic products and glassware. In order to avoid recontamination of plasmid DNA after initial endotoxin removal, it is recommended to use only new plastic products that are certified pyrogen-free or endotoxin free. Endotoxin-free or pyrogen-free plastic products are available from many different suppliers. Endotoxins strongly adhere to the glassware and are difficult to remove completely during washing. Standard laboratory autoclaving methods have little or no effect on endotoxin levels. In addition, if the pressure cooker has been previously used for bacteria, the glassware will be widely contaminated by endotoxin molecules. Heating the glassware at 180 ° C overnight can destroy any additional endotoxin molecules. It is important not to purify endotoxin-free DNA by using endotoxin-containing reagents, all of which require endotoxin free.