protein purification slides
TRANSCRIPT
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?
Results Number of fluorescent colonies counted:
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.
Conclusion
Protein PurificationStudent Training
Objective:
To extract and separate fluorescent proteins in E.coli from other cellular debris (membrane, other proteins, DNA, RNA, etc.)
Transformation
Bacteria now express cloned fluorescent protein…
Bacterial chromosome
Allow bacteria to grow for 1-3 days on plate with ampicillin.
Plasmid Uptake of foreign DNA, often a circular plasmid
Bacterial chromosome
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
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.
Purify a specific protein from over 4,000 naturally occurring E. coli gene products.
How do you purify proteins?
Organisms produce thousands of different proteins, each having a different function…
HormonesHair and Nails
Structural Support
Enzymes
Muscle Contraction
Antibodies
Receptors, membrane channels
Nutrient Storage
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)
Protein Structure
Protein Structure1° = amino acids
2° = basic structure(hydrogen bonds)
3° = 3D structure
4° = interactionof subunits
Why Purify Proteins?
Research
• to make vaccines • to treat disorders
• to understand structure
Medicine
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
How do you purify proteins?
Fluorescent proteins are just one of thousands of
proteins in the cell!
Fluorescent Protein
Purify a specific protein from over 4,000 naturally occurring E. coli gene products.
How do you purify proteins?
How do we purify proteins? (3)
1. Break open the cells in 2 ways
- Adding Lysozyme
- Snap Freeze
0. Scrape cells into the tube with TE Buffer
Lysozyme is a naturally occurring enzymethat is used to break open cells.
Lysozyme
How do we purify proteins? (3)2. Centrifuge the cells to separate the heavy cell material from lighter cell material supernatant
pellet
How do we purify proteins? (3)3. Separate the fluorescent protein from other light cellular debris using column chromatography
3a. Mix supernatant with nickel beads
3b. Pass the supernatant and nickel bead mixture through the column and into a waste tube
3c. Add elution buffer to column and collect solution in a new tube
How do the nickel beads work?
his-his-his-h
is-his-h
is
His- tag: a chain of histidine amino acids
How do the nickel beads work?
Ni2+
Ni2+
Ni2+
Ni2+Ni2+
Ni2+
Ni2+
The his-tag on the fluorescent protein and nickel bind like 2 magnets
How does the elution buffer work?
Elution buffer contains a molecule called imidizole that has a stronger attraction to the nickel beads
Ni2+
Ni2+
Ni2+
Ni2+
Ni2+
Ni2+
How do we purify proteins?
Finished! Now you have a pure sample containing only fluorescent proteins
Why purify proteins?In Research: In Medicine:
To characterize protein structure, function and interactions
To create vaccines from recombinant proteins such as insulin and factor 8
Tricky Parts of Lab Scraping cells into tube
Using the correct buffer solution