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44 Cards in this Set
- Front
- Back
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What type of chromatography was used to isolate lysozyme from egg white? |
Cation-exchange chromatography |
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What does lysozyme do? |
Attacks cell wall of bacteria--> breaks B-1,4-glycosidic bonds between NAG and NAM |
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Who discovered lysozyme? What else did said individual discover? |
Alexander Fleming, penicillin |
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Is lysozyme a drug? |
No, it's too big to travel between cells |
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Lysozyme was the first enzyme to... |
...have it's structure solved!! |
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Brian Matthews |
- induced mutations on lysozyme - attempted to create new active site w/ new molecule-shaped pockets |
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Where can lysozyme be found? |
Blood, immune system, tears, food, human milk |
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Isoelectric point of lysozyme |
11 |
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Why do charged molecules in a solution migrate? |
Cause of electric field |
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What are the three types of electrophoresis and which did we conduct? |
i) Tube gel
ii) Slab gel** iii) Flat bed |
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Ohm's Law |
I = E/R where... I= current (amperes) E= energy (volts) R= resistance (ohms) |
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Formula for Power |
p = EI where... p= power (watts) E= energy (volts) I= current (amperes) |
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Amphoteric compounds |
- proteins with acidic and basic residues - nucleic acids are NOT amphoteric |
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Isoelectric Point |
pH where protein has no net charge |
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Above a protein's pI... |
-ve charge, migrates towards anode |
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Below a protein's pI... |
+ve charge, migrates towards cathode |
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Why is maintaining a constant temperature in electrophoresis important? |
- Polymerization is exothermic - If not regulated--> irregularities in pore size - Avoid denaturing proteins - Heat conducted away from slab--> move faster in center (smile effect) - Run at 0dc or 25dc - Lower buffer needs to cover tube or slab |
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What are some support matrixes? |
Polyacrylamide and agarose gel |
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Support Matrix |
- inhibits convection caused by heating - must be electrically neutral (prevent electroosmosis) |
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Agarose support matrix |
- Separates macromolecules (nucleic acids) |
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Polyacrylamide suport matrix |
- Separates most proteins & small oligonucleotides |
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Electroosmosis |
Flow of solvent towards one of the electrodes |
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Agarose Gels |
- polysaccharide from agar - dissolve in boiling lq, gels at 40dc - higher conc = smaller pores - fragile--> H-bonds and hydrophobic bonds |
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Polyacrylamide Gels |
Tougher--> covalently linked by crosslinker |
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Preparing gel |
- prepare in absence of O2 (purge w/ inert gas or vacuum) - add thin layer of water to prevent meniscus |
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Pore size can be adjusted in which two ways? |
- % acrylamide: increased % decreases pore size - % crosslinker: 5% creates smallest pores |
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Pore Gradient Gel |
- Larger pores at top, smaller at bottom |
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2 Types of Polymerization |
i) Chemical: use ammonium persulfate & TEMED ii) Photochemical: riboflavin & TEMED - long wavelength UV |
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Tricky part of polymerization |
- generates heat and too much can cause inconsistencies - time controlled by initiator catalyst (20-60 min) |
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Three types of analysis for SDS-PAGE |
i) Staining ii) Autoradiography iii) Blotting |
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Analysis of Gel: Staining |
- Most common - Coomassie Blue - Photographic amplification system - Row transition metals - Scan/Photograph - Ethidium bromide--> Nucleic Acid |
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Analysis of Gel: Autoradiography |
- detect radioactive samples - dry gel and place X-ray film on top - exposed--> radioactive bands |
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Analysis of Gel: Blotting |
- transfer proteins from slab to membrane - transfer by capillary or southern blotting (SLOW) - Vacuum blotting--> memb. & gel on vacuum chamber |
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Continuous Buffer System |
- one separating gel - same buffer in tanks & gel |
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Discontinuous Buffer System |
- 2 buffer solution - Gels layers - Greater resolution |
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Two types of buffer systems |
i) Continuous ii) Discontinuous |
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SDS-PAGE (what does it stand for??) |
Sodium dodecylsulfate- polyacrylamide gel electrophoresis |
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What does SDS do? |
Denatures proteins and gives them negative charge |
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SDS-PAGE- linear relationship exists between... |
log10(MW) and Rf |
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Rf formula |
= distance from top of gel to protein/ distance from top of gel to solvent front |
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Isoelectric focusing |
- protein separation based on pI - samples on 1 gel - horizontal w/ polyacrylamide or agarose |
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2 stages of isoelectric focusing |
i) Create pH gradient- nonrestrictive gel is polymerized ii) Protein migration- once at pI stops migrating |
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How to read pH gradient to assign pI |
- run standard protein w/ known pI's OR - dummy is sliced. Slices are dissolved in salt. Then test pH. |
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2D Electrophoresis |
- 1D run in tube gel & grafted horizontally onto polymerized slab gel for SDS-PAGE - O'Farrell (1975) - Used to separate complex mixtures into more components |
