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.
                

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.)

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)

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

Chemical pulldown

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

Purification of DNA and RNA binding substances