BCHM 309 1st Edition Lecture 9 Outline of Last Lecture I Tertiary Structure II Favorable Residues A Secondary Structure B Tertiary Structure III Protein Folding A Protein Denaturing B Denaturing Agents Outline of Current Lecture I Protein Purification A Protein Purification Methods II Gel Filtration Chromatography III Ion Exchange Chromatography A Monitoring Progress IV Gel Electrophoresis A Visualizing the Proteins Current Lecture A protein can be purified in order to be studied Purifying will separate out individual enzymes for studying This can be done by crystallography gene cloning through the use of antibodies and through protein sequencing One method to study proteins is through chromatography This involves separating a mixture s components into two phases a mobile and stationary phase This can be done through gel filtration chromatography or ion exchange chromatography This process is gentle and helps to preserve the protein s structure This is important as chromatography is often used to study its function which is dictated by structure Electrophoresis is another method used This separates a mixture s components based on their charge and or size The size of a protein is usually given as its molecular weight in Daltons This can also be expressed as a sum of all the amino acid component s masses This process can be done by polyacrylamide gel electrophoresis or isoelectric focusing The downside to this method is that the protein becomes denatured This method is used when the goal is to evaluate purity Gel filtration chromatography is sometimes referred to as size exclusion chromatography The mobile phase of this process contains a buffer which the proteins are soluble in A column of beads make up the stationary phase These beads have holes in them larger than most of the proteins in the solution Smaller substances flow through the holes in the beads and elute or exit slowly The larger molecules will pass through the column quickly because they can t go through the beads Ion exchange chromatography also contains a buffer in the mobile phase The stationary phase contains beads as well Instead though the beads are coated in a positive or negative charge that is covalently attached to them These are in the form of chloride or sodium counter ions A specific type is called anion exchange chromatography The beads only carry a positive charge Therefore negatively charged molecules will bind to the beads displacing the counter ions The repelled positively charged substances are able to quickly elute The substances that are bound to the beads can be eluted through two methods The first is to add a buffer that contains a gradient of counter ions in increasing concentrations A change in pH will also cause the same effect During the course of performing a chromatography the UV absorbance is monitored The aromatic amino acids in the proteins will have an absorbance around 280 nanometers A protein that is also an enzyme can have its enzymatic activity monitored This will help to show how pure a protein is Normally the purification process has to be repeated multiple times Polyacrylamide gel electrophoresis is known as PAGE Sodium dodecyl sulfate PAGE SDS PAGE is a type of this method often used Proteins contain a great diversity in size shape and charge This process coats the protein with a negative charge This causes the proteins to all behave as negatively charged rods This type of electrophoresis heats the proteins in a mixture of SDS and mercaptoethanol The mercaptoethanol breaks disulfide bonds The SDS binds to the hydrophobic regions imparting the negative charge This also completely unfolds the proteins allowing them to behave in a more uniform manner All structure except for the primary is lost All enzymatic activity is also gone Once this is done the electrophoresis process continues as the sample is added to a gel An electrical current applied to the gel causes the proteins to move As all the proteins are negatively charged they will migrate downwards to the positive end Larger proteins will move slower an end up more towards the top After the current is applied a dye can be added to visualize the protein bands
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