研究目的
To study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence of BSA-Au(III) complexes and to understand the origin of the red fluorescence.
研究成果
The study demonstrates that the emergence of red fluorescence in BSA-Au(III) complexes is correlated with pH-induced conformational changes in BSA. Multiple binding sites for Au(III) in BSA are identified, including the Asparagine fragment and Cys-34. The necessity of Cys-Cys disulfide bonds for red fluorescence production is highlighted. The protocols can be applied to study interactions of other proteins with metal cations.
研究不足
The technique requires precise control of pH and temperature to avoid protein denaturation. The process of red fluorescence emergence is slow and requires days to reach maximum intensity at room temperature. High acidity from excess gold can denature the protein.
1:Experimental Design and Method Selection:
The study employs spectroscopic and protein chemistry techniques to analyze the BSA-Au(III) complexes.
2:Sample Selection and Data Sources:
Bovine Serum Albumin (BSA) and gold (III) chloride trihydrate (chloroauric acid) are used.
3:List of Experimental Equipment and Materials:
Includes HPLC grade water, BSA, chloroauric acid, NaOH, copper (II) chloride dihydrate, nickel (II) chloride hexahydrate, N-ethylmaleimide (NEM), phosphate buffered saline (PBS), urea, ammonium bicarbonate (NH4HCO3), tris(2-carboxyethyl)phosphine (TCEP).
4:Experimental Procedures and Operational Workflow:
Involves synthesis of BSA-Au(III) complex, BSA-Cu(II)-Au(III), BSA-Ni(II)-Au(III), [Cys34-capped-BSA]-Au(III), and [all-thiol-capped-BSA]-Au(III) under controlled pH and temperature conditions.
5:Data Analysis Methods:
Fluorescence spectra and excitation-emission maps (EEM) are analyzed to understand the binding sites and fluorescence patterns.
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HPLC grade water
Used as a solvent in the synthesis of BSA-Au(III) complexes.
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gold (III) chloride trihydrate
Source of Au(III) for binding to BSA.
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copper (II) chloride dihydrate
Used to study competitive binding with Au(III) to BSA.
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nickel (II) chloride hexahydrate
Used to study competitive binding with Au(III) to BSA.
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N-ethylmaleimide (NEM)
Thiol capping agent used to modify cysteine residues in BSA.
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phosphate buffered saline (PBS)
Used as a buffer in the synthesis of [Cys34-capped-BSA]-Au(III).
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urea
Used in the preparation of [all-thiol-capped-BSA]-Au(III).
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ammonium bicarbonate (NH4HCO3)
Used in the preparation of [all-thiol-capped-BSA]-Au(III).
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tris(2-carboxyethyl)phosphine (TCEP)
Used to cleave disulfide bonds in BSA.
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