Protein Expression and Purification Series - Bio-Rad

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APPENDIX I GLOSSARY Protein Expression and Purification Series Chromatography – A process for separating complex mixtures of proteins or other molecules. In the case of column liquid chromatography, separation is accomplished by passing a liquid mixture over a column containing a solid matrix. The properties of the matrix can be tailored to allow the selective separation of one kind of molecule from another. Properties include hydrophobicity, molecular size, and charge. Column – A plastic or glass cylinder that is densely filled (“packed”) with small, porous spheres or beads. Column bed – The volume of beads packed within the chromatography column. Column volume – (CV), the volume of buffer equal to the volume of resin packed in a chromatography column. Dalton – One Dalton equals the mass of a hydrogen atom, which is 1.66 x 10 -24 grams. A DNA kilobase pair has a mass of approximately 660 kD. Decant – Gently removing liquid or buffer from a column or other vessel so as not to disturb the resin or sediment bed. ΔOD/min – Change in optical density, or measured absorbance, per minute. For this series, it is used in calculating the activity of an enzyme. Denaturation – Process of disrupting a protein’s structure. DHF – Dihydrofolate or dihydrofolic acid. DHFR – Dihydrofolate reductase. Disulfide bond – S—S (sulfide—sulfide) bond between amino acids in a polypeptide chain; contributes to tertiary and quaternary structure of proteins. Dithiothreitol – (DTT) is a reducing agent used to break the disulfide bonds of proteins, thus disrupting the tertiary and quaternary structure of the protein. It linearizes and prepares the protein for electrophoresis. DNase – Enzyme that specifically breaks down DNA. Electrophoresis – Means ”to carry with electricity.” It is the migration of charged molecules in an electric field toward the electrode with the opposite charge. Eluate – The solution of buffer and biomolecules from elution. Elute – The removal of a bound molecule from a chromatography resin. Elution buffer – The chromatography buffer containing chemicals used for the removal of a bound molecule from a chromatography resin. Exclusion limit – The upper size limit for molecules that cannot penetrate the pores of the porous beads. See Fractionation range. Fraction – A tube that contains material that has flowed through the chromatography column. Multiple tubes or fractions are collected during each chromatography run. 242 Appendix I: Glossary
Protein Expression and Purification Series Fractionation range – For size exclusion chromatography gels, the fractionation range is the range of molecular weights that will enter the gel. A fractionation range of 1,000–6,000 kD will have pores large enough for molecules in that size range to pass through. Molecules larger than 6,000 kD will be excluded from entering the gel. The fractionation range is sometimes referred to as the “exclusion limit.” Frit – Bed support of the chromatography column. Gel electrophoresis – Technique used to separate, or sieve, molecules that carry electric charges. The molecules separate from each other according to the different rates at which they migrate through an electric field set up in a gel that is soaked in a chemical solution. Glycoslyation – An enzymatic process that adds glycans, or sugars, to a protein or other organic molecule. Glycosylation is known to aid in protein folding. GST-tag – Glutathione-s-transferase, an enzyme that binds to the substrate glutathione, is a small amino acid sequence (27 kD mw) that is added to the sequence of a recombinant protein. Glutathione is bound to chromatography resin and thus used to purify proteins with the GST-tag. GST is also added to recombinant proteins to aid in solubility. His-tag – A series of histidine residues (usually 6) fused to a protein that aids protein purification because of its strong binding to nickel (IMAC) columns. Also known as a “polyhistidine tag.” Hydrophilic – A molecule that has a strong affinity for water, “water loving.” Hydrophobic – A molecule that has a strong dislike for or is insoluble in water, “water fearing.” Hydrophobic interaction chromatography – A chromatography method that separates molecules based on their level of hydrophobicity. IMAC – Immobilized Metal Affinity Chromatography; a chromatography method where the affinity of histidines to metals, such as nickel, is used to purify proteins tagged with polyhistidine sequences. Inclusion body – Aggregated and precipitated expressed proteins found inside bacteria induced to make high levels of recombinant protein. Insoluble – The parts of the cell that are not dissolved in water or buffer. Ion exchange chromatography – A chromatography method where the charge of the molecule is exploited to bind to oppositely charged chromatography media. Isoelectric point – (pI) The pH at which a molecule has a net charge of 0. Laemmli sample buffer – The first, and most common, sample buffer used for protein electrophoresis. First described in 1970, this buffer consists of 62.5 mM Tris buffer to maintain pH conducive to electrophoresis; 10% glycerol to increase density of the protein so that it stays sunk in the gel well, 2% SDS to equalize the protein charge; 5% DTT (or BME) can be added to reduce disulfide bonds in the protein; and 0.01% bromophenol blue, which gives the sample color. Ligand – A molecule, such as an antibody, enzyme, or protein tag, with specific affinity for another molecule. Loading buffer – (Equilibration buffer) The buffer used to add sample to a chromatography column. The loading buffer is formulated to exploit properties of the biomolecule of interest for the particular chromatography resin and allows the biomolecule to bind to the resin. Appendix I: Glossary 243 APPENDIX I GLOSSARY
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Biotechnology Explorer TM Protein E
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Protein Expression and Purification
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CHAPTER 1 BACKGROUND Protein Expres
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CHAPTER 2 FOCUS QUESTIONS Protein E
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CHAPTER 3A ADVANCE PREP CENTRIFUGAT
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CHAPTER 3B ADVANCE PREP INSTRUMENTA
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CHAPTER 4 11 ml CULTURE PROTOCOL Pr
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CHAPTER 5 100 ml CULTURE PROTOCOL P
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CHAPTER 6 HANDPACKING COLUMNs Prote
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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
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Page 192 and 193: CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 202 and 203: CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
Page 210 and 211: APPENDIX A TIPS AND HELPFUL HINTS P
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Page 214 and 215: APPENDIX B RESULTS ANALYSIS Protein
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Page 218 and 219: APPENDIX C RCF TO RPM CONVERSTION P
Page 220 and 221: APPENDIX D SETTING UP THE SMARTSPEC
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Page 224 and 225: APPENDIX E MINI-PROTEAN GEL BOX SET
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Page 230 and 231: APPENDIX G BIOMANUFACTURING Protein
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Page 234 and 235: APPENDIX H FOCUS QUESTIONS INSTRUCT
Page 244 and 245: APPENDIX I GLOSSARY Protein Express
Page 246 and 247: APPENDIX J REFERENCES, LEGAL NOTES
Page 250: Life Science Group Bulletin 1665067
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Protein Expression and Purification Series - Bio-Rad

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