Protein Purification Slides
- 1. Warm up! Sit next to your lab partner from yesterday Put on a lab coat and gloves. Take out your lab notebook. Title: Bacterial Transformation Purpose: To change the phenotype (physical traits) of e.coli by changing the geneotype (DNA or genes) Method: See ScienceBridge Protocol Results: “Wait for Quanina to explain” Take a look at your results. Did you see what you expected?
- 2. Results Number of fluorescent colonies counted: Conclusion If you saw colonies growing that were not fluorescent, explain how this could have happened? If you saw no colonies growing, how could this have happened? If your results were unexpected, explain how this could have happened.
- 4. Objective: To extract and separate fluorescent proteins in E.coli from other cellular debris (membrane, other proteins, DNA, RNA, etc.)
- 5. Transformation Bacterial Plasmid Uptake of foreign DNA, chromosome often a circular plasmid Allow bacteria to grow for 1-3 Bacterial days on plate with ampicillin. chromosome Bacteria now express cloned fluorescent protein…
- 6. Warm-up Sit next to your lab partner from yesterday. Take out your PD and reading packet annotations. Swap PDs and annotate. Repeat until 3 people have read and annotated your PD. Do this without talking
- 7. SLCs Yay! Take out a sheet of paper. Write down 3 things you are proud that you have accomplished this semester. Write down 2 things you want to improve on this semester. You can talk about any class you are taking this semester or you can talk about the semester overall.
- 8. How do you purify proteins? Purify a specific protein from over 4,000 naturally occurring E. coli gene products.
- 9. Organisms produce thousands of different proteins, each having a different function… Hair and Nails Hormones Structural Support Muscle Contraction Receptors, Enzymes membrane channels Antibodies Nutrient Storage
- 10. Proteins… …are created by living organisms (DNA → RNA → PROTEIN → trait) …have unique structures that determine function (insulin, cobratoxin, fluorescence) …can be isolated from living things (humans, cobras, jellies) …can be studied and modified by humans (fluorescent proteins)
- 12. Protein Structure 1° = amino acids 2° = basic structure (hydrogen bonds) 3° = 3D structure 4° = interaction of subunits
- 13. Why Purify Proteins? Research Medicine • to understand structure • to make vaccines • to treat disorders
- 14. Why Purify Proteins? Pancreas → Identify Cells → Isolate Gene → Insert Gene → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein Insulin for → Human Use diabetics
- 15. Why Purify Proteins? Pancreas → Identify Cells → Isolate Gene → Insert Gene → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein Insulin for → Human Use diabetics
- 16. Why Purify Proteins? Pancreas → Identify Cells → Isolate Gene → Insert Gene → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein Insulin for → Human Use diabetics
- 17. Why Purify Proteins? Pancreas → Identify Cells → Isolate Gene → Insert Gene → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein Insulin for → Human Use diabetics
- 18. Why Purify Proteins? Pancreas → Identify Cells → Isolate Gene → Insert Gene → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein Insulin for → Human Use diabetics
- 19. Why Purify Proteins? Pancreas → Identify Cells → Isolate Gene → Insert Gene → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein Insulin for → Human Use diabetics
- 20. Why Purify Proteins? Pancreas → Identify Cells → Isolate Gene → Insert Gene → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein Insulin for → Human Use diabetics
- 21. Why Purify Proteins? Pancreas → Identify Cells → Isolate Gene → Insert Gene → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein Insulin for → Human Use diabetics
- 22. How do you purify proteins? Fluorescent proteins are just one of thousands of proteins in the cell! Fluorescent Protein
- 23. How do you purify proteins? Purify a specific protein from over 4,000 naturally occurring E. coli gene products.
- 24. How do we purify proteins? (3) 0. Scrape cells into the tube with TE Buffer 1. Break open the cells in 2 ways - Adding Lysozyme - Snap Freeze
- 25. Lysozyme Lysozyme is a naturally occurring enzyme that is used to break open cells.
- 26. How do we purify proteins? (3) 2. Centrifuge the cells to separate the heavy cell material from lighter cell material supernatant pellet
- 27. How do we purify proteins? (3) 3. Separate the fluorescent protein from other light cellular debris using column chromatography 3a. Mix supernatant with 3b. Pass the supernatant 3c. Add elution nickel beads and nickel bead mixture buffer to column and through the column and collect solution in a into a waste tube new tube
- 28. How do the nickel beads work? His- tag: a chain of histidine amino acids
- 29. How do the nickel beads work? The his-tag on the fluorescent protein and nickel bind like 2 magnets Ni2+ Ni2+ Ni2+ Ni2+ Ni2+ Ni2+ Ni2+
- 30. How does the elution buffer work? Elution buffer contains a Ni2+ molecule called imidizole that has a stronger Ni2+ attraction to the nickel Ni2+ beads Ni2+ Ni2+ Ni2 +
- 31. How do we purify proteins? Finished! Now you have a pure sample containing only fluorescent proteins
- 32. Why purify proteins? In Research: In Medicine: To create vaccines from recombinant proteins such To characterize protein as insulin and factor 8 structure, function and interactions
- 33. Tricky Parts of Lab Scraping cells into tube Using the correct buffer solution
Editor's Notes
- #9: Like your body, the E. coli bacterial cells that we will be using have many different proteins. From the over 4,000 proteins on an E. coli cell, we will be separating out just the specific protein that we are interested in looking at. The protein we will be purifying is the fluorescent protein.A bacterial cell is full of stuff. This includes DNA, RNA, lipids, and many different types of proteins. If we only want to separate out the fluorescent protein, how are we going to do that?
- #14: Recombinant vaccines expressed in bacteria or yeast to produce large quantities of a single viral or bacterial protein. -purified protein injected into the patient -patient's immune system makes antibodies to the disease agent's protein, -patient protected from natural disease. Advantages of the recombinant vaccine technology are that there is virtually no chance of the host becoming ill from the agent, since it is just a single protein, not the organism itself.
- #24: Like your body, the E. coli bacterial cells that we will be using have many different proteins. From the over 4,000 proteins on an E. coli cell, we will be separating out just the specific protein that we are interested in looking at. The protein we will be purifying is the fluorescent protein.A bacterial cell is full of stuff. This includes DNA, RNA, lipids, and many different types of proteins. If we only want to separate out the fluorescent protein, how are we going to do that?
- #25: LysozymeIn our saliva, tears, spleen, lung, kidney High concentration in chicken egg-white (our source of lysozyme).Lysozyme was discovered accidentally in 1922 by Alexander Fleming by accident. Nasal drippings in the petri dish with bacterial culture, killing the bacterial cells. Viruses uselysozymeto break into the host bacterial cell allowing it to inject its DNA. SNAP FREEZEThis technique involves freezing and then thawing the material.Causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing.