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The Hydroxyl-functionalized Magnetic Particles for Purification of Glycan-binding Proteins

Author: SunXiuZuo
Tutor: LiZuoï¼›ChenChao
School: Northwestern University
Course: Microbiology
Keywords: Hydroxylation of magnetic particles Reducing sugar Sugar-binding proteins Separation and purification Differential expression profile
CLC: Q513-3
Type: Master's thesis
Year: 2009
Downloads: 139
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Abstract


Background: The sugar-binding proteins (glycan binding proteins, GBPs) and a sugar chain of glycoprotein or glycolipid sugar chain interactions regulate cell identification, signal transduction, cell endocytosis and intracellular transport of substances, cell growth, differentiation, and apoptosis, outside of pathogenic microorganisms infections of some of the most basic and most important biological functions. Home and abroad to take a combination of affinity chromatography and ion exchange chromatography separation and extraction of GBPs mainly, but due to the strong non-specific adsorption of this method, time-consuming and complicated to operate, the target protein concentration shortcomings, is not conducive to low abundance degree of protein research. Based the GBPs separation technology shortcomings, we have developed a new technology based on magnetic particle separation and purification GBPs can be highly efficient, rapid and easy separation and purification of GBPs. Objective: To establish functionalized magnetic particles as a carrier covalently coupled of reducing sugars chain of methods, the use of linked sugar chains of magnetic particles functionalized sugar binding protein separation and purification of biological protein samples, and the sugar binding protein research proteomics preliminary. Methods: functional magnetic microparticle carrier covalently coupled to mannose, glucose and cellobiose sugar, to optimize the immobilization and separation and extraction of sugar-binding protein of the conditions of the three sugars. Healthy human serum, the serum of patients with Kashin-Beck and serum of patients with liver cancer sugar binding protein separation and extraction, two-dimensional electrophoresis and protein, the two-dimensional electrophoresis gel analysis software through 2DImageMaster background subtraction, the identification and matching of protein spots . Comparing patients with healthy human serum sugar binding protein expression differences. Results: reducing sugar chain with four functions of magnetic particles (Fe 3 O 4 magnetic particles, the epoxidation magnetic particles, magnetic particles hydrazine and hydroxylation of magnetic particles ) coupled to compare the experimental results show that the hydroxylation of the magnetic particles on the capacity and stability of the reducing sugar chain coupled with the other three magnetic particulates. Followed hydroxylated on the magnetic particles modified conditions were optimized to determine the best chemical modification time, 4 - hydroxy-benzoyl hydrazine concentration and the reaction temperature. Through optimization of the sugar chain and the optimal coupling of the magnetic fine particles of the hydroxylated buffer system, the pH of the coupling buffer, temperature conditions of the coupling, the sugar chain and the coupling amount of the magnetic fine particles of the hydroxylated optimal. The combined buffer system and elution system of complex sugar magnetic particles (linked sugar chains of magnetic particles) stability, the results show that both the complex stability, there was no significant impact. Make sure the 1% SDS elution buffer as a sugar binding protein. Healthy human serum using this method, the the large joints patient serum and serum of patients with liver cancer sugar binding protein separation and purification by SDS-PAGE and silver staining shows the similarities and differences of patients with healthy human serum carbohydrate-binding protein, and combination of 2D electrophoresis image analysis, respectively, to establish a healthy human serum and serum of patients with Kashin-Beck difference spectra of healthy people with the serum of patients with liver cancer in sugar-binding proteins. Conclusion: hydroxylation of magnetic fine particles as a carrier can be achieved a variety of reducing sugars immobilized modified. More reducing sugar chains under the same conditions can be effectively coupled. The sugar binding protein in the complex sample of the hydroxylated magnetic particulates a fixed reducing sugar chain separation and purification, this method can reduce the complexity of the protein component, can be applied proteomics research. Differences of healthy human serum and serum carbohydrate-binding protein expression profiling combined with two-dimensional electrophoresis.

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CLC: > Biological Sciences > Biochemistry > Protein > Binding proteins
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