Magnetic recognition system
A magnetic and ferromagnetic technology, applied in the field of magnetic recognition labels, can solve the problems of protein orientation not being optimal, deterioration, etc.
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Embodiment 1
[0156] Example 1 - Design and Manufacturing of Fusion Proteins
[0157] To illustrate the present invention, fusion proteins were designed using a commercially available murine anti-fibronectin antibody. Fusion proteins of anti-fibronectin scFv linked genetically to MT2 or ferritin via short flexible linkers were made. This example details the construction, characterization and isolation of the fusion protein.
[0158] The design of the anti-fibronectin ferritin or MT2 fusion protein is based on combining the V from the mouse anti-fibronectin antibody H and V L Genes are cloned into vectors. Both genes are linked by a short, flexible linker consisting of small uncharged amino acids. immediately after V L At the 3' end of the gene, another short flexible linker was ligated to either the ferritin gene or the MT2 gene. Both fusion proteins have hexahistidine regions for purification on nickel columns. Fusion protein translation was terminated at the inserted stop codon at ...
Embodiment 2
[0199] Embodiment 2——SPR analysis
[0200] Anti-fibronectin ferritin and MT2 fusion protein inclusion body preparations were used in surface plasmon resonance (SPR) analysis using a SensiQ instrument (ICX Nomadics).
[0201] For these experiments, fibronectin peptides were coupled to the carboxyl chip surface. The fusion protein preparation was then flowed through the chip and binding (K a ) and dissociation (K d )dynamics.
[0202] Fusion protein analysis samples
[0203] Six samples of each fusion protein at different concentrations of 0.0013 μM to 0.133 μM in running buffer were prepared, as shown in Table 2 and Table 3 below.
[0204] Table 2 - Metallothionein fusion proteins 75kDa:
[0205] 400 μL 10 μg / mL (0.133 μM) was obtained from 40 μL 100 μg / mL 75kDa / 360 μL running buffer, then
[0206] μM FP
μg / mL FP
10μg / mLFP (μL)
Running buffer (μL)
0.0013
0.1
20(of 1μg / mL)
180
0.0065
0.5
10
190
...
Embodiment 3
[0221] Example 3——Magnetized ferritin
[0222] Ferritin usually contains hydrated iron(III) oxide. To generate paramagnetic ferritin, magnetite (Fe 3 o 4 ) to replace these ions. The method used in this experiment involves adding iron ions to apoferritin and oxidizing these ions under controlled conditions.
[0223] Material
[0224] ●Reverse osmosis water (RO water)
[0225] 50mM N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO) buffer (pH8.6) (Sigma A6659)
[0226] ●0.1M sodium acetate buffer (pH 4.5)
[0227] ●Phosphate-buffered saline (PBS), 10mM phosphate, 140mM NaCl (pH 7.4)
[0228] ●Trimethylamine-N-oxide (TMA) (Sigma 317594)
[0229] ●0.1M ammonium ferrous (II) sulfate
[0230] ●Horse spleen apoferritin (Sigma A3641)
[0231] method
[0232] Trimethylamine-N-oxide (TMA) was heated to 80 °C (30 min) in an oven to remove Me 3 N, then cooled to room temperature. 114mg TMA was added to 15mL RO water to make a 0.07M solution. Iron ...
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