Using the “chemical pull-down method,” which is a method for purifying target proteins using FG beads immobilized with a drug as a ligand, we analyze the functions and reaction mechanisms of molecules in vivo at the molecular level. FG beads are best at chemical biology because they can immobilize various kinds of compounds and have very little non-specific adsorption.
The site “1” and site “2” of the compound involve to the activity and are inactivated by immobilization on FG beads, so immobilization at the site “3” is required.
After immobilizing the compound (drug) on the FG beads, the target protein is recovered (bound).
After binding the compound (drug) and the target protein, the complex of “compound + target protein” is recovered by FG beads. It is necessary to introduce azide, alkyne, biotin, tag, etc.
※Please scroll horizontally.
Difficulty of immobilization | Functional group at the site of immobilization (compound side) |
FG beads to use | |
---|---|---|---|
Low | Direct method | NH2 | NHS beadsCOOH beads |
COOH | NH2 beadsOH beads | ||
Azide | Alkyne beads | ||
Alkyne (Ethynyl) | Azide beads | ||
OH(Phenolic) | Epoxy beads | ||
OH | COOH beads | ||
Indirect method | Azide | Alkyne beads | |
High | Alkyne (Ethynyl) | Azide beads |
Experiments will not work if the amount of compound immobilization is high or low. First of all, it is important to carry out experiments with the amount of preparation under 4 conditions and find the optimum immobilization conditions for each compound. |
By performing competitive inhibition and drug elution, it is also possible to narrow down the binding protein band and evaluate the specificity of the binding protein.
Isolation of drug target protein ‐ Methotrexate
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Isolation of drug target protein ‐ Bisindolylmaleimide
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Isolation of drug target protein ‐ Vorinostat
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Isolation of drug target protein – Biotinylated MTX
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Purification of proteins binding Bisindolylmaleimide X
This study is the collaboration with a pharmaceutical company.
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Purification of target proteins for kinase inhibitor Bisindolylmaleimide Ⅷ
This study is a cooperative research with Medical ProteoScope Co., Ltd./p>
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