Magnetic substance-biosubstance complex structure, peptide fragment capable of linking to magnetic substance and gene therefor, and process for producing the complex structure
a biosubstance and complex structure technology, applied in the field of peptide fragments capable of linking to magnetic substances, can solve problems such as conversion or denaturation of biosubstances, and achieve the effect of high reproducibility
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reference example 1
Preparation of Magnetic Particles
[0068] An aqueous solution containing ferrous hydroxide was prepared by adding, to an aqueous ferrous sulfate solution, a sodium hydroxide solution in an amount of 1.0-1.1 equivalent to the ferrous ion. Air was blown into this aqueous solution by keeping the pH of the aqueous solution at about 8 to cause oxidation reaction at 80-90° C. to obtain a liquid slurry for formation of seed crystals.
[0069] To this liquid slurry, was added an aqueous ferrous sulfate solution in an amount of 0.9-1.2 equivalent to the above added alkali (the sodium of the above added sodium hydroxide), and air is blown therein to proceed an oxidation reaction by keeping the pH at about 8. The formed magnetic iron oxide particles after the oxidation reaction was collected by filtration, washed, and dried. The particulate iron oxide in an aggregating state was crushed to obtain magnetic particles (1) having an average particle size of 0.10 μm.
example 1
Formation of Peptide Structure Capable of Linking to Magnetic substance
1. Preparation of Liquid Suspension of Magnetic Particles
[0070] To 5 mg of magnetic particle (1) prepared in Reference Example 1, was added 1 mL of a TBS buffer (50 mM tris-HCl (pH 7.5), 150 mM NaCl) to form a suspension. The suspension was centrifuged at 10,000 rpm (9300 g) for 5 minutes, and the supernatant was eliminated. The precipitate was suspended in 1 mL of acetone. The suspension was again centrifuged and the supernatant was eliminated under the same conditions as above. Further to the precipitate, was added 1 mL of a TBS-0.1T buffer (50 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween-20) to form a suspension. The suspension was centrifuged under the above conditions, and the supernatant was eliminated. This operation was repeated two more times. The obtained precipitate was suspended in 1 mL of a TBS-0.1T buffer. To 10 μL of the suspension, was added 990 μL of the TBS-0.1T buffer to prepare a magnetic ...
example 2
Evaluation of Linkage of Magnet-Linking Peptide Structure to Magnetic substance
[0074] A 5 μg portion of the magnetic fine particles prepared in Reference Example 1 was suspended in 100 μL of a TBS-0.1T buffer (50 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20). Thereto was added 10 μL of a solution containing 2×10 pfu of the phage having a peptide of the amino acid sequence SEQ ID NO:15. The mixture was agitated at room temperature (25° C.) for 30 minutes to cause linking reaction. Further it was suspended in TBS-0.5T buffer (50 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Tween 20), and was recovered by magnetic force. This treatment was repeated 10 times to release and wash off the non-adsorbed and non-specifically adsorbed phage.
[0075] According to the method described in Nature, 405, 665-668 (2000), the recovered magnetic fine particles containing specifically adsorbed phage were allowed to react with an anti-fd phage antibody-biotin complex (produced by Sigma Co.) and streptavid...
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