| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
I developed a new specific extraction method for capturing small molecule-binding proteins from biological fluids by the cleavable affinity gel. This gel has the small molecule ligand immobilized onto the agarose gel via the disulfide linker, and the captured proteins can be extracted to the supernatant as the ligand-protein complex by reductive cleavage reaction without any conformational change of proteins.
In the model experiment, this method was applied to extraction of deoxycholate-binding proteins in mouse ascites containing anti-deoxycholate monoclonal antibody. In SDS-PAGE, three protein bands were observed in the extraction fraction, and the mass spectrometric analysis and database search following in-gel tryptic digestion indicated that they were serum albumin and heavy chain and light chain of anti-deoxycholate monoclonal antibody.
Next, I tried to extract the chenodeoxycholate-binding proteins in the rat brain tissue. The cytoplasmic fraction of cerebrum, midbrain, cerebellum, brainstem, hippocampus, and pituitarium were separately mixed to the chenodeoxycholate-immobilized cleavable affinity gel, and the captured proteins were analyzed by SDS-PAGE. All tissues contain three protein bands in the extraction fraction. After in-gel tryptic digestion and analysis by MALDI-TOF MS, they were identified as α-, β-tubulin, β-actin, and 14-3-3 protein by the database search. Moreover, two abundant protein bands were observed in the extraction fraction from pituitarium, and identified as serum albumin and growth hormone by MALDI-TOF MS and database search. Serum albumin is one of the carrier proteins of bile acids, and plays an important role in bile acid transport in blood stream. The extraction of serum albumin indicated that this extraction method was useful for specifically capturing small molecule-binding proteins from biological fluids.
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