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#2 Filled in Skeleton Notes

#2 Filled in Skeleton Notes - Hogan Chem 130 Lecture notes...

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Hogan, Chem 130 Lecture notes Chapter 5: Protein purification I. Characteristics of Proteins A. Every Protein has a unique primary sequence. Hence, every protein has several unique characteristics B. As biochemists, we are interested in understanding the chemical and biological properties of proteins. Consequently, we need methods to selectively isolate proteins of interest from the rest of the cellular milieu. 1. Approx. # of proteins in an E. coli cell : 3,000 2. Approx. # of proteins in a human cell: ~30,000 II. Precautions A. Factors that influence protein stability: 1. Temperature – must be kept cold for protins to remain intact 2. pH – buffer must be @ physoligical pH (or @ pH favorable to protein) 3. Ionic strength – [high] of salts will denature protein, so salts must be right. III. Methods of cell disruption A. There are a number of ways to prepare a protein extract from prokaryotic and eukaryotic cells. Homogenization – breaking up cells 1. Mechanical methods: a. Blender – violently lyses cells and intracellular organelles b. Sonication – using sound, sometimes saves organelles c. Potter-Elvejhem homogenizer scrape tissue in thick walled test tube – also saves organelles 2. Enzymatic methods: a. Lysozyme – and enzyme that digests cell walls 3. Next, differential centrifugation –separate out proteins from intact organelles 4. Next, technique called “salting out” done 1 1. Size – based on primary sequences 2. Shape – (globular or fibril) 3. Charges (net) dictated by type of AA’s 4. Solubility also affected by AA’s 5. Subunit compositions (tertiary quaternary) Reason we investigate proteins: -want to find properties of specific known proteins -Find protein partners in a functional AA sequence All techniques typically used in combination!
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Hogan, Chem 130 Lecture notes a. Use ammonium sulfate and find [salts] in which protein will elude from a column B. Maintaining the stability of proteins once cells have been lysed 1. Be sure to keep your proteins happy: Carefully formulate a buffer: -Temp, pH, [salt] Maintain everything **Temp to purify protein must be kept below 10 C C. Monitoring for the presence of a protein 1. Enzyme Activity Assay: ??? 2. Immunological: ??? III. Protein Purification Methods – Based on the following properties: 1. Solubility 2. Charge 3. Size 4. Natural binding affinity A. Purification based on Solubility 1. The effects of salt concentration on solubility -don’t know [salt] – tall trial and error at first -[salts] = low increase protein solubility -[salts] = high decrease protein solubility *basically, the more ammonium sulfate you add, the less and less soluble protein becomes, until it comes out of solution and is separated – at this [salts] or [ammonium sulfate], protein can be purified. 2.
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#2 Filled in Skeleton Notes - Hogan Chem 130 Lecture notes...

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