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Protein Analysis
Protein Analysis
Prof. Conlons Lecture Unit 9 - Molecular Medicine 22nd Feb 2003 Week 2
4MedStudents.com
Main Steps in Protein Analysis 1. Extraction of proteins. 2. Purification of the protein. 3. Structural Characterization of the protein.
1. Protein Extraction
Involves the efficient extraction of the protein and peptide in a biologically active form from tissue. During this process, inactivation of proteolytic enzymes is required. Why ? Not to cause degradation of the proteins contained in the tissue. Extraction of protein: homogenize it in a physiological buffer (0.05M NaPO4, PH 7.4) containing proteolytic enzyme inhibitors (EDTA, Pepstatin) Extraction of peptides: boil the tissue with 1M Acetic Acid for 5 min homogenize tissue in ethanol / 0.1MHCl (ratio 3:1) at 0C. This will burst open the vesicles to release the peptides in solution. 4
2. Protein Purification
Proteins can be purified according to certain properties they possess. These properties allow us to employ different techniques in purifying proteins.
Protein Property Solubility Molecular Size Molecular Charge Hydrophobicity Biological Activity
Technique Differential Precipitation Gel Filtration Chromatography Ion Exchange Chromatography Reversed Phase HPLC Affinity Chromatography
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Protein Purification
A. Differential Precipitation
Precipitation: the process of formation of a solid that was previously held in solution. The solid is separated from the solution. When NH4 SO4 or polyethylene glycol are added to a protein solution, a precipitate forms and it can be separated from the solution after centrifugation. If the concentrations of NH4 SO4 or polyethylene glycol are increased, more precipitate forms.
Protein Purification
Protein Purification
Protein Purification
Protein Purification
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Protein Purification
Protein Purification
E. Affinity Chromatography
It is the most powerful means of purifying proteins. It takes the advantage of the high affinity of many proteins to specific chemical groups or molecules. Example:
Concavalin A is a glucose binding protein. It can be purified by passing it through a column that has special beads. These beads have glucose attached to them. The glucose binding protein binds to the beads and other proteins dont. The protein can be released from the beads by increasing the concentration of glucose. This will displace the glucose binding protein from the beads.
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Protein Purification
E. Affinity Chromatography
Other proteins can be separated by this method based on their affinity for specific groups or compounds. Examples:
Antigen Antibody Substrate Concavalin A Hormone Antibody Antigen Enzyme Glycoprotein Binding Protein/Receptor
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3. Protein Characterization
Characterization of proteins and peptides involves three different processes: 1.
2.
3.
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Protein Characterization
Protein Characterization
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Protein Characterization
Cleavage Point
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Protein Characterization
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Protein Characterization
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