Excellent tool for chemical biology
In pharmaceutical research, it is extremely important to perform isolate and identify the protein that is the target of a drug (compound) in vivo. However in the past, isolation and identification of the drug target protein were extremely difficult, and were a problem which required large amounts of time and effort. In order to overcome this problem, through joint research with Professor Hiroshi Handa at the Tokyo Institute of Technology, Tamagawa Seiki has developed the new nanomagnetic particles "FG beads®" and the automated screening system "Target Angler".
FG beads®, are approximately 0.2 μm in diameter and are composed of a plurality ferrite particles coated with a unique polymer called poly-GMA (glycidyl methacrylate). FG beads®, that are manufactured by using this original technology, are used as a carrier for affinity purification and provide characteristics that are superior to conventional carriers, allowing one-step purification of the target proteins. The development of an automated screening system, that magnetically separates and disperses FG beads®, makes it possible to automate the affinity purification process. Concequently, the simultaneous process of multiple samples and time shortening of the process are enabled.
FG beads can capture a variety of substances, including chemicals (drugs), proteins, and DNA. From the 9 types of FG beads, select the type with the optimal surface modification according to the functional group of the substance you want to bind. Because FG beads are resistant to various organic solvents, they are able to bind a variety of ligands. (Avoid using streptavidin beads or other protein-binding FG beads in organic solvents.) The beads with ligands immobilized can be used for affinity purification of the target biological substance.
Protein purification (affinity purification) using FG beads is performed in 3 stages:
binding, washing, and elution.
During the binding process, the beads immobilized ligands are mixed with the crude cell extract, and the proteins with affinity for the ligands bind to the beads. During the washing process, the proteins that have bound non-specifically to beads (not the proteins bound specifically to the ligands) are washed off. During the elution process, the specifically-bound proteins are separated from the ligands and recovered. At each of these processes, magnets are used to separate immobilized beads from the crude cell extract, washing buffer, or elution buffer.
Affinity purification of MTX binding proteins
Immobilization of MTX on commercial magnetic beads was done in the same manner as in the case of FG beads.
The nano size of FG beads gives them excellent dispersibility. As a result, magnetic separation in an organic solvent may be difficult, and it is necessary to recover the FG beads by centrifugal separation.
Centrifugal separation causes the FG beads to agglutinate strongly, making it difficult to disperse them. Ordinarily a manual dispersion method or ultrasound would be used to disperse the beads. Although ultrasound separates the beads easily, caution is required due to the possibility of damaging the proteins. Therefore in general it is recommended that ultrasound be used when binding low molecular weight compounds, and that manual dispersion be used when binding proteins. The manual method involves resting the bottom of the micro tube in a plastic test tube stand and moving it roughly to disperse the beads. Depending on the type of micro tube, when using the manual method to disperse the beads, the bottom of the tube may crack or leakage from the lid may occur. Use a micro tube that is strong and has a lid with a tight fit. We recommend the use of cap locks.
When centrifugal separation is performed, heavy proteins and insoluble proteins are also precipitated, raising the level of the background. Because FG beads have high dispersibility, magnetic separation requires time (in some cases 5 minutes or longer). However using magnetic separation avoids the risk of intrusion by these impurities and provides clear results with a low background level.
If dispersion is insufficient following the FG beads washing process after the binding reaction with the proteins, there is the possibility of impurities remaining inside the bead clusters. Therefore it is necessary to disperse the beads well. Use the manual method to disperse the beads. (With ultrasound, there is the risk that the proteins will be damaged.)
|Application Note 1:||Isolation of drug target protein|
|Application Note 2:||Cell separation, THP-1|
|Application Note 3:||Isolation of drug target protein
Protein Kinase inhibitors (Bisindolylmareimide)
|Application Note 4:||Isolation of drug target protein
Histone Deacetylase Inhibitor (Vorinostat)
|Application Note 5:||Immunoprecipitation|
Purification of target protein of Thalidomide (elucidation of the teratogenic mechanism)
CRBN (Cereblon) and DDB1 were isolated from human cell extract using thalidomide fixed beads. As a result, the teratogenic mechanism of thalidomide was elucidated.
T. Ito et al., Science 327 (2010) 1345
Purification of novel target protein of MTX (methotrexate)
When MTX was fixed via different site, a novel protein was purified and identified as deoxycytidine kinase (dCK). As a result, a possible mechanism of synergistic effect between MTX and ara-C on malignant lymphoma was proposed.
H. Uga et al., Mol. Pharmacol. 70 (2006) 1832
Purification of target proteins of Capsaicin
Prohibitin 1 and prohibitin 2 were isolated from human myeloid leukemia NB4 cell extract using capsaicin derivative (Cap-NH2) fixed beads. As a result, the apoptosis induction mechanism of capsaicin was elucidated.
Elucidation of mechanism of enteropathogenic E. coli enfection
EspB is a protein of enteropathogenic E. coli (EPEC) essential for infection in humans, Myosin was isolated from human cell extract using EspB fixed beads. As a result, the mechanism of EPEC infection was elucidated.
Y. lizumi et al., Cell Host & Microbe. 2 (2007) 383
The Latest Reference Documents (Tamagawa Seiki Co., Ltd. web site)
|E001||Screening by using ligand immobilized beads||101KB|
|E003||Immobilization of ligands (compounds with phenol groups or NH2 groups) on epoxy beads||90KB|
|E004||Immobilization of ligands (carboxylic compounds) on OH beads||67KB|
|E005||Immobilization of ligands (carboxylic compounds) on NH2 beads||95KB|
|E008||Immobilization of ligands (compounds with NH2 groups) on COOH beads||133KB|
|E014||Immobilization of ligands (compounds with NH2 groups) on NHS beads||88KB|
|E108||Immobilization of Ligand on Streptavidin beads||89KB|
|E109||Immobilization of ligands (alkyne structure compounds) on azide beads using click chemistry reaction||74KB|
|E201||Quantifying the amount of ligand immobilization by HPLC (High Performance Liquid Chromatography)||2,345KB|
The protocols above are for immobilization of 2.5 mg of a ligand. For smaller scale, click the link here to get protocols.
Immunoprecipitation, Protein-protein interaction
|E101||Immobilization of proteins on COOH beads||89KB|
|E102||Immobilization of His-Tag proteins on Ts beads||96KB|
|E105||Immobilization of Antibodies or Proteins on NHS beads||80KB|
|E106||Immobilization of Antibodies or Proteins on Epoxy Beads||78KB|
|E107||Direct Quantification of Immobilized Proteins (Antibodies)||102KB|
|E108||Immobilization of Ligand on Streptavidin beads||93KB|
|E110||Immobilization of antibodies on Protein A beads and Protein G beads||134KB|
Purification of DNA and RNA binding substances
|E001||Screening by using ligand immobilized beads||101KB|
|E301||Immobilization of double strand DNA on Plain beads||137KB|