The document provides instructions for purifying fluorescent proteins expressed in E. coli, including breaking open the cells, separating cell debris via centrifugation, and using nickel beads and column chromatography to isolate the fluorescent proteins from other proteins in the supernatant. Key steps involve lysing the cells with lysozyme, binding the fluorescent proteins to nickel beads via their histidine tags, and eluting the purified proteins from the column using imidazole buffer.

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.

3. Protein Purification
Student Training

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)

11. Protein Structure

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