Products

High performance magnetic nanoparticle (FG beads)

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 200 nm in diameter that are composed of multiple ferrite particles coated with a special polymer called poly-GMA (glycidyl methacrylate). The FG beads that are manufactured using this original technology are used as materices for affinity purification and provide characteristics that are superior to conventional materices, allowing one-step purification of the drug target protein. The development of an automated screening system that magnetically separates and disperses the FG beads makes it possible to automate the affinity purification process, process multiple samples simultaneously, and shorten the time required.

Features

  • Extremely low non-specific binding:
    poly-GMA (Glycidyl methacrylate) coated magnetic nanoparticles
  • Excellent recovery of target proteins:
    200 nm particles have a large surface area and a high dispersibility
  • High stability in organic solvents:
    various compounds can be immobilized on the beads

Product Information

Various Magnetic Beads

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.

Linkers and Functional Groups

Purification process

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.

Comparison with other magnetic beads

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.

Applications

Application Note (PDF)

Application Note 1: Isolation of drug target proteinpdf
Application Note 2: Cell separation, THP-1pdf
Application Note 3: Isolation of drug target protein
Protein Kinase inhibitors (Bisindolylmareimide)pdf
Application Note 4: Isolation of drug target protein
Histone Deacetylase Inhibitor (Vorinostat)pdf
Application Note 5: Immunoprecipitationpdf

Application Examples

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

Protocols

Protocol for Specific Application

Chemical pulldown

E001 Screening by using ligand immobilized beads pdf
259KB
E003 Immobilization of ligands (compounds with phenol groups or NH2 groups) on epoxy beads pdf
111KB
E004 Immobilization of ligands (carboxylic compounds) on OH beads pdf
78KB
E005 Immobilization of ligands (carboxylic compounds) on NH2 beads pdf
97KB
E008 Immobilization of ligands (compounds with NH2 groups) on COOH beads pdf
139KB
E012 Competitive inhibition pdf
261KB
E013 Drug elution pdf
260KB
E014 Immobilization of ligands (compounds with NH2 groups) on NHS beads pdf
75KB
E015 Immobilization of MTX aminated derivatives on NHS beads pdf
66KB
E108 Immobilization of Ligand on Streptavidin beads pdf
102KB
E109 Immobilization of ligands (alkyne structure compounds) on azide beads using click chemistry reaction pdf
65KB
E201 Quantifying the amount of ligand immobilization by HPLC (High Performance Liquid Chromatography) pdf
1,329KB

Immunoprecipitation, Protein-protein interaction

E011 Immunoprecipitation pdf
77KB
E101 Immobilization of proteins on COOH beads pdf
83KB
E102 Immobilization of His-Tag proteins on Ts beads pdf
89KB
E105 Immobilization of Antibodies or Proteins on NHS beads pdf
23KB
E106 Immobilization of Antibodies or Proteins on Epoxy Beads pdf
65KB
E107 Direct Quantification of Immobilized Proteins (Antibodies) pdf
107KB
E108 Immobilization of Ligand on Streptavidin beads pdf
102KB
E110 Immobilization of antibodies on Protein A beads and Protein G beads pdf
103KB

Purification of DNA and RNA binding substances

E001 Screening by using ligand immobilized beads pdf
259KB
E301 Immobilization of double strand DNA on Plain beads pdf
143KB

Tips; Background Reduction

Immobilization

  1. Perform centrifugal separation.
    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.
  2. Disperse the beads well.
    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.

Dispersion by sonication

Screening

  1. Perform magnetic separation
    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.
  2. Disperse the beads well
    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.)

Manual dispersion method

References


The Latest Reference Documents
(Tamagawa Seiki Co., Ltd. web site)

  1. M, Watanabe et al., Oncogenesis, 6, e311 (2017)
  2. Y, Nishiya et al., Bioorg. Med. Chem. Lett., 27, 834 (2017)
  3. L. Jia et al., Biochem. Eng. J., 114, 268 (2016)
  4. T, Taniguchi et al., Cell Death Dis., 7, e2211 (2016)
  5. Jaiyam Sharma et al., Sensing and Bio-Sensing Research 9, 7?12 (2016)
  6. Y. kabe et al., Nature Communications, 7:11030 (2016)
  7. Y. Soeda et al., Nature Communications, 6:10216 (2015).

Downloads

Ordering Information

FG beads

Product Name Storage Product No. PKG Size Price
Plain beads 4°C TAS8848 N1010 10 mg e-Nacalai
Linker beads (Epoxy beads) 4°C TAS8848 N1110 5 mg
NH2 beads 4°C TAS8848 N1130 5 mg
COOH beads 4°C TAS8848 N1140 5 mg
NHS beads -20°C TAS8848 N1141 5 mg
Azide beads 4°C TAS8848 N1160 5 mg
Streptavidin beads 4°C TAS8848 N1170 5 mg
NeutrAvidin™ beads 4°C TAS8848 N1171 5 mg
Protein A beads 4°C TAS8848 N1172 5 mg
Protein G beads 4°C TAS8848 N1173 5 mg

Others

Product Name Storage Product No. PKG Size Price
Magnetic Stand (for 1.5 ml tube) Room Temp. TA4899N12 1 ea e-Nacalai
Magnetic Stand (for 15 ml tube) Room Temp. TA4899N20 1 ea
Magnetic Stand (for 50 ml tube) Room Temp. TA4899N30 1 ea
MTX derivatives 4°C TAS8849N101 0.1 mg

(Storage) RT: Room temperature, A: Cool and dark, R: Refrigerator, F: Freeze