Nacalai USA - Innovations for Life Sciences

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What are FG beads?

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 beads 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 beadsmakes it possible to automate the affinity purification process, process multiple samples simultaneously, and shorten the time required.

Features

  • Extremely low non-specific bindingpoly-GMA (Glycidyl methacrylate) coated magnetic nanoparticles.
  • Excellent recovery of target proteins200 nm particles have a large surface area and a high dispersibility
  • High stability in organic solventsvarious compounds can be immobilized on the beads.
 

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.

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.
                

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.

 

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.)

Protocol for Specific Application

Chemical pulldown

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

Immunoprecipitation, Protein-protein interaction

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

Purification of DNA and RNA binding substances

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

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

 

 

 

Reference

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

  1. S. Sakamoto et al., Chem. Rec. 9 (2009) 66
  2. K. Nishio et al., Colloids Surfaces. B. 64 (2008) 162
  3. T. Ito et al., Science 327 (2010) 1345
  4. H. Uga et al., Mol. Pharmacol. 70 (2006) 1832
  5. C. Kuramori et al., BIochem. Biophys. Res. Commun. 379 (2009) 519
  6. Y. lizumi et al., Cell Host & Microbe. 2 (2007) 383
  7. M. Ueda, Chem. Lett. 41 (2012) 658
  8. N. Maekawa et al., Biomed. Chromatogr. 25 (2011) 466
  9. A. Tominaga et al., Genes to Cells 15 (2010) 595
  10. E. Yoshihara et al., Nature Communications (2010)
  11. K. Kume et al., Genes to Cells 15 (2010) 339
  12. M. Hiramoto et al., Biomed. Chromatogr. 24 (2010) 606
  13. E. Suzuki et al., Bioorg. Med. Chem. 17 (2009) 6188
  14. M. Hatakeyama et al., J. Magn. Magn. Mater. 321 (2009) 1364
  15. J. Kang et al., Genes Dev. 23 (2009) 208
  16. Y. Hase et al., Biochem. Biophys. Res. Commun. 336 (2008) 66
  17. M. Azuma et al., PLoS ONE (2008)
  18. K. Nishio et al., J. Magn. Magn. Mater. 310 (2007) 2408
  19. M. Hasegawa et al., Bioorg. Med. Chem. Lett. 16 (2006) 158

Ordering Information

FG beads
Product Cat.No. Storage Quantity Price(US$)  
Plain beads TAS8848 N1010 4°C 10 mg 280.00 Buy
Linker beads (Epoxy beads) TAS8848 N1110 4°C 5 mg 240.00 Buy
NH2 beads TAS8848 N1130 4°C 5 mg 320.00 Buy
COOH beads TAS8848 N1140 4°C 5 mg 320.00 Buy
NHS beads TAS8848 N1141 -20°C 5 mg 350.00 Buy
Azide beads TAS8848 N1160 4°C 5 mg 350.00 Buy
Streptavidin beads TAS8848 N1170 4°C 5 mg 350.00 Buy
NeutrAvidin™ beads TAS8848 N1171 4°C 5 mg 350.00 Buy
Protein A beads TAS8848 N1172 4°C 5 mg 150.00 Buy
Protein G beads TAS8848 N1173 4°C 5 mg 150.00 Buy
Others
Product Cat.No. Storage Quantity Price(US$)  
Magnetic Stand (for 1.5 ml tube) TA4899N12 Room Temp. 1 ea 800.00 Buy
Magnetic Stand (for 15 ml tube) TA4899N20 Room Temp. 1 ea 600.00 Buy
Magnetic Stand (for 50 ml tube) TA4899N30 Room Temp. 1 ea 700.00 Buy
MTX derivatives TAS8849N101 4°C 0.1 mg 50.00 Buy