2. Lisa Yoneda Mira Mesa High School Teacher Background:
Biology to AP Biology
Biotechnology
Background:
BS Molecular Biology
MA Educational Technology
LSSI alumni (2nd year)
Coordinate UCSD ScienceBridge Kits

3. Laboratory 6 Part B: Getting what we need-Protein
LSSI Alum
Lisa Yoneda, Biotechnology Program, Mira Mesa HS

4. Safety General Lab Safety Guidelines
Use laboratory coats, safety glasses and gloves as appropriate
Avoid restrictive clothing and open-toed shoes
No eating or drinking in the lab
Make sure that students are familiar with the operating instructions and safety precautions before they use any of the lab equipment
Check all MSDS (Material Safety Data Sheets) for all chemicals and reagents in the lab before preparing and running the lab
Wash hands at the conclusion of the lab.
Lab Safety Guidelines for lab 6
When using potentially bio-hazardous materials work in a sanitary manner and treat all waste as a potential biohazard
Dispose of pipette tips and all other materials that came in contact with bacteria in the biohazard bag provided
Any item potentially contaminated by bacteria should be treated with 70% ethanol or another acceptable disinfectant

5. Understanding Columns
Day 2 Background
Understanding Columns

6. Warm Up
Many medicines today are proteins. Biotechnology companies make these medicines the same way you transformed the bacteria to make rfp.
However, the protein is in the bacteria, so how do we get our protein out without destroying it’s properties?
What effects a proteins ability to do it’s job?

7. Understanding purification Methods:
Hydrophobicity is often used to help separate molecules.
Hydrophobic: ‘fears water’ : oil, wax, fats
Hydrophilic: ‘loves water’ : salt, sugar
Proteins have both hydrophobic and hydrophilic parts
Hydrophilic regions point outward
Hydrophobic regions point inward
(reversed for membrane proteins)

8. With over 1,000 different proteins in the cell how can we isolate rfp?
We used an expression vector (araC)
The cells are making more rfp than any other protein
Column chromatography:
Hydrophobic region of rfp is used to separate from other proteins

9. RFP Structure
Yi Shen ; Tiffany Lai and Robert E. Campbell "Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications", Neurophoton. 2(3), (Jun 19, 2015). ;

10. RFP Structure High Salt Solution Low Salt Solution
Hydrophobic regions bend outward
Low Salt Solution
Hydrophobic regions fold inside

11. Column Structure
Column
Stop Cock
Matrix
Solvents

12. Column Structure Column Stop Cock
Glass (or plastic) tube that holds matrix
Stop Cock
Regulates flow of solvents
In line: allows solvents to flow
Perpendicular: blocks solvents- stops flow
(May not have)

13. Column Structure Matrix Other names
Resin
Stationary phase
Coated beads that can bind and release your molecule under different conditions
Note:
Must be kept in liquid at all times
Keep 2 mm (~ ½ pinky nail) of liquid above the resin at all times
Never disturb resin
Add solvents slowly and down side of the column- no clouds

14. Column Structure Purpose Solvents/Buffers Equilibration Binding Wash
Elution
Storage
Prepares matrix for binding to molecule of interest
Usually mixed with supernatant before adding to matrix- prepares rfp for attaching to matrix/resin
Removes other molecules from matrix- allows rfp to stay bound
Rfp detaches and flows out of matrix
Keeps resin stable when not in use (will not be sent in your kit)

15. Binding Buffer High Salt Buffer Notes: Mix with rfp supernatant
Rfp exposes inner hydrophobic amino acids
Rfp and hydrophobic proteins will bind to resin
Hydrophillic proteins will flow through
Notes:
Flow through = clear = waste
Ideally add solvent slowly- rfp will bind in tight ring- increasing final concentration

16. Wash Buffer Medium Salt Buffer Notes:
Allows some refolding of proteins- but not rfp
Moderately hydrophobic proteins release and flow through
RFP will stay bound to resin
Notes:
Flow through = clear = waste
Gravity flow can be slow

17. Elution Buffer Rfp will refold- covering hydrophobic amino acids
Rfp releases and flows through
Note:
Add slowly and all rfp will release and flow through together
Only catch pink/red solution with clean tube
Clear solution = buffer only = waste

18. Column Animation
Interactive Animation: Protein Purification Using Hydrophobic Interaction Chromatography (HIC)

19. Part B (Day 2)
Step 3: Centrifuge
Step 4: Column

20. Outline Part B Protocol (prelab)
Notes/Changes
Flow chart of each main step
Draw with labels
Be sure to note where the rfp should be for each step
Enough details that can use your notes to do the lab
Amounts
Time
Name of solutions

21. Step 3: Centrifuge
What will happen when we centrifuge the lysed cells?
A) Centrifuge 5 min (high speed)
Supernatant – rfp & cytoplasm proteins
Pellet – Large cell pieces

22. Step 3: Centrifuge B) 200 uL Supernatant into NEW tube (no pellet)
C) 200 uL BB
A) Centrifuge 5 min (high speed)
Supernatant – rfp & cytoplasm proteins
Pellet – Large cell pieces

23. Column Prep Setup columns Arm/finger clamp Ring stand Waste container
Column tip should be just above waste container
Predrain (if stop cock attached)
Note: ALWAYS leave ~2 mm of solution above resin
This is when you add next buffer
Never drain resin dry
If no stop cock- don’t pre drain (wait to drain until ready to add next solution)
start
Ready for next buffer

24. Step 4: Run Column A) Drain CEB
Always keep ~ 2 mm of solution above resin
If no stop cock, be ready with next solution
start
Ready for next buffer

25. Step 4: Run Column Add buffers slowly down side of column- no clouds
B) Add BB/rfp mixture (400 uL = all) & run through column
Where will rfp end up?
rfp
Clear waste
Add buffers slowly down side of column- no clouds

26. Step 4: Run Column
B) Add BB/rfp mixture (400 uL = all) & run through column
C) Add 1 mL WB & run through column
Where will rfp end up?
rfp
rfp
Clear waste
Clear waste
Add next buffer when draining buffer is still above the resin (~ 2mm)

27. Step 4: Run Column Only collect red liquid- let clear flow into waste
B) Add BB/rfp mixture (400 uL = all) & run through column
D) Add 1 mL EB & run through column (do 2X = 2 mL total)
C) Add 1 mL WB & run through column
rfp
rfp WB
Where will rfp end up?
Clear waste
Clear waste
Only collect red liquid- let clear flow into waste

28. Step 4: Column Storage E) Reset/Store Column
Run 2 mL of CEB through column
(Final class- add extra 1 mL on top of resin)
Cap tightly (top & bottom)
Start/storage level

29. Gravity columns can run slow
Can use plunger to increase column flow rate
Never pull up on plunger while attached to column
Needs tight seal- columns can be warped into oval shape
Check if clamp is too tight
Gently squeeze column into round shape
Make sure you still have drops, not a steady stream coming from column when using

30. Making Plungers Can make plunger to increase column flow rate
#2 stopper- 1 hole
10 mL syringe
Attach together by cutting 1 mL disposable pipet

31. Part B (Day 2) Overview Step 3: Centrifuge
5 min: Separate cytoplasm proteins from cell walls and membranes
200 uL supernatant into clean tube
Add 200 uL binding buffer (BB)

32. Part B (Day 2) Overview Step 4: Run Column
Column prep (1 group member- 1st class only)
Setup Column Apparatus
400 uL Binding Buffer/rfp mixture
rfp stays in column
1 mL Wash Buffer (WB)
2 mL Elution Buffer (EB)
Only catch red drops in clean microfuge tube
2 mL CEB Buffer (reset column)

33. Protein Purification Procedure Where is rfp? Cytoplasm inside the cell
Centrifuge - transformed liquid culture
Lyse cells- overnight incubation
Centrifuge – lysed cells
Column – Purification of rfp
Cytoplasm inside the cell
Liquid portion (cells cut open)
Supernatant (liquid)
Attach to resin in column and then release

34. Use your flowgram to complete Lab 6 Part B
Complete Part B (Day 2)
Use your flowgram to complete Lab 6 Part B
Step 3: Centrifuge
Step 4: Run Column

35. Part B (Day 2) Teacher Prep Notes
Columns run slow
Can setup columns ahead of time
Students Centrifuge & setup column at same time
Slow columns
Make sure
stop cock open
Caps (top & bottom) are removed
Unclog by resettling resin
Apply pressure with plungers

36. Part A (Day 1) Teacher Timing Notes
Can pre aliquot 1000 uL rfp into student tubes (1/group)
If pressed for time can do 2/group and then have them combine (add EB to 1 pellet-mix and transfer into second pelleted tube)
Can do Part B overview or column setup during centrifuge times
Centrifuge loud (need microphone or put in back room)

37. Part B (Day 2) Teacher Timing Notes
Columns run slow
Can setup columns ahead of time
Block schedule ideal
Apply pressure with plungers
Students Centrifuge & setup column at same time
Pre-centrifuge overnight incubation
Remove frozen 1 period before

38. Video Links for Lab 6 Lab 6 Purification Help Column Help
Lysing the cells (quantities differ)
Column Protein Purification Overview
Eluting rfp
Column Help
Unclogging columns
Column basic setup & mistakes

39. Reagent Only Lab Prep & Aliqouting Guidelines
Reagents/Supplies
Aliquot
Storage Temp
Notes
1 flask of 250 mL of LB/Amp/Ara broth (OR use plate from Lab 5 to inoculate/Kit
N/A
4°C
Please return flask
10 Resin packed columns RT
Please return columns
Already in Column Equilibration Buffer
150 mLs of Elution Buffer/ kit (EB)
2 mL/group
150 mLs of Wash Buffer/kit (WB)
1 mL/group
150 mLs of Column Equilibration Buffer/kit (CEB)
50 mLs of Binding Buffer/kit (BB)
200 uL/group
150 mls of Ethanol/kit (EtOH)
Storage Buffer- may not be in the kit. If not, leave Column Equilibration Buffer in the columns.
1.6 mls of Lysis Buff/class (LyB)
150ul/group
Equipment/Supplies
10 Student boxes with the following:
1 p20 micropipette microfuge rack
1 p200 micropipette bag of microfuge tubes
1 p1000 micropipette box of refillable tips (2 ul-200 ul)
1 waste bucket
4 Mini centrifuges
1 Tabletop centrifuge
10 Ring stands with clamps
10 Boxes of p1000 tips
10 Pressure Syringes for Columns