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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)
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
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?
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.
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?
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.