Rainbow Protein Purification Lab：彩虹蛋白纯化实验室.ppt

Proteins… …are created by living organisms (Central Dogma: DNA=RNA=PROTEIN=trait) 2) …have unique structures that determine function (insulin, cobratoxin, fluorescence) 3) …can be isolated from living things (purification) (humans, cobra, jellies) 4) …can be studied and modified by humans (fluorescent proteins) Protein Structure Fluorescent Proteins (Green) Fluorescent Protein Red, Green, and Rainbow Proteins Column Chromatography-Step 2 Proteins… …are created by living organisms (Central Dogma: DNA=RNA=PROTEIN=trait) 2) …have unique structures that determine function (insulin, cobratoxin, fluorescence) 3) …can be isolated from living things (purification) (humans, cobra, jellies) 4) …can be studied and modified by humans (fluorescent proteins) Materials Checklist—Day 2 Plate with transformed bacteria, expressing fluorescent protein Mini centrifuge tube Disposable Plastic Pipet Cell Scraper TE buffer solution (each group needs 2 mls) Sharpie Marker Waste Container Lysozyme from frozen tubes (teachers re-hydrate and keep on ice) Dry Ice and Ethanol (see teacher) Materials Checklist—Day 3 (or Day 2) Defrosted lysed cell solution 150μl nickel beads (in microfuge tube labeled “Ni”) on ice Pasteur pipet Cotton Straw or stick Disposable Pipets (2) Mini centrifuge Elution buffer (in a microtube labeled “EB”) Waste Container Engineered Fluorescent Proteins Day 2 (or 3): Column Chromatography 6. Place a clean microtube below column. With a NEW pipet, release fluorescent protein from Ni beads by adding “elution buffer” consisting of an imidazole solution. Watch as fluorescent protein slowly leaves Ni Beads and flows into microtube. From GFP: From RFP: 1. Green 4. Cherry 2. Blue 5. Tangerine 3. Grape 6. Yellow We will use the plates made at the Teacher Workshop for the Student workshop later. Have students give examples of human proteins within these categories (some examples may be keratin,