Transcript Part B (Day 2)

Lisa Yoneda
• Mira Mesa High School Teacher
– Biology to AP Biology
– Biotechnology
• Background:
– BS Molecular Biology
– MA Educational Technology
– LSSI alumni (2nd year)
– Coordinate UCSD ScienceBridge Kits
www.amgenbiotechexperience.com
LABORATORY 6 PART B: GETTING
WHAT WE NEED-PROTEIN
LSSI Alum
Lisa Yoneda, Biotechnology Program, Mira Mesa HS
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
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Day 2 Background
Understanding Columns
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?
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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)
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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
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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), 031203 (Jun 19,
2015). ;http://dx.doi.org/10.1117/1.NPh.2.3.031203
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RFP Structure
• High Salt Solution
– Hydrophobic regions bend outward
• Low Salt Solution
– Hydrophobic regions fold inside
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Column Structure
•
•
•
•
Column
Stop Cock
Matrix
Solvents
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Column Structure
• Column
– Glass (or plastic) tube that holds
matrix
• Stop Cock
– Regulates flow of solvents
• In line: allows solvents to flow
• Perpendicular: blocks solventsstops flow
– (May not have)
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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
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Column Structure
Solvents/Buffers
Purpose
• Equilibration • Prepares matrix for binding to molecule of interest
• Binding
• Wash
• Elution
• Storage
• 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)
Binding Buffer
• High Salt Buffer
– 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
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Wash Buffer
• Medium Salt Buffer
– Allows some refolding of proteinsbut 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
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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
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Column Animation
• Interactive Animation: Protein Purification
Using Hydrophobic Interaction
Chromatography (HIC)
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PART B (DAY 2)
Step 3: Centrifuge
Step 4: Column
Outline Part B Protocol (prelab)
Protocol
• 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
Notes/Changes
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
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Step 3: Centrifuge
B) 200 uL Supernatant into
NEW tube (no pellet)
A) Centrifuge 5 min
(high speed)
Supernatant – rfp &
cytoplasm proteins
Pellet – Large cell
pieces
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C) 200 uL BB
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)
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start
Ready
for
next
buffer
Step 4: Run Column
• A) Drain CEB
– Always keep ~ 2 mm of solution
above resin
– If no stop cock, be ready with
next solution
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start
Ready
for
next
buffer
Step 4: Run Column
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
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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
Clear waste
rfp
Clear waste
• Add next buffer when draining buffer is still
above the resin (~ 2mm)
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Step 4: Run Column
B) Add BB/rfp
mixture (400 uL
= all) & run
through column
rfp
C) Add 1 mL WB
& run through
column
D) Add 1 mL EB
& run through
column (do 2X =
2 mL total)
rfp
WB
Where will rfp
end up?
Clear waste
Clear waste
• Only collect red liquid- let clear flow into
waste
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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)
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Start/storage
level
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
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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
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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)
www.amgenbiotechexperience.com
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)
• rfp stays in column
– 2 mL Elution Buffer (EB)
• Only catch red drops in clean microfuge tube
– 2 mL CEB Buffer (reset column)
www.amgenbiotechexperience.com
Protein Purification
Procedure
Where is rfp?
• Cytoplasm inside the cell
1.
Centrifuge - transformed
liquid culture
2.
Lyse cells- overnight
incubation
• Liquid portion (cells cut
open)
3.
Centrifuge – lysed cells
• Supernatant (liquid)
4.
Column – Purification of rfp
• Attach to resin in column
and then release
Complete Part B (Day 2)
Use your flowgram to complete Lab 6 Part B
• Step 3: Centrifuge
• Step 4: Run Column
www.amgenbiotechexperience.com
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
www.amgenbiotechexperience.com
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)
www.amgenbiotechexperience.com
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
www.amgenbiotechexperience.com
Video Links for Lab 6
• Lab 6 Purification Help
– Lysing the cells (quantities differ)
– Column Protein Purification Overview
– Eluting rfp
• Column Help
– Unclogging columns
– Column basic setup & mistakes
www.amgenbiotechexperience.com
Reagent Only Lab Prep & Aliqouting
Guidelines
Reagents/Supplies
1 flask of 250 mL of LB/Amp/Ara broth (OR
use plate from Lab 5 to inoculate/Kit
10 Resin packed columns
Aliquot
N/A
Storage Temp
4°C
N/A
RT
150 mLs of Elution Buffer/ kit (EB)
2 mL/group
RT
150 mLs of Wash Buffer/kit (WB)
1 mL/group
RT
150 mLs of Column Equilibration Buffer/kit
(CEB)
50 mLs of Binding Buffer/kit (BB)
2 mL/group
RT
200 uL/group
RT
150 mls of Ethanol/kit (EtOH)
N/A
RT
1.6 mls of Lysis Buff/class (LyB)
150ul/group
4°C
Equipment/Supplies
10 Student boxes with the following:
1 p20 micropipette
1 p200 micropipette
1 p1000 micropipette
1 waste bucket
4 Mini centrifuges
1 Tabletop centrifuge
10 Ring stands with clamps
10 Boxes of p1000 tips
10 Pressure Syringes for Columns
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Notes
Please return flask
Please return columns
Already in Column Equilibration Buffer
Storage Buffer- may not be in the kit. If not, leave
Column Equilibration Buffer in the columns.
1 microfuge rack
1 bag of microfuge tubes
1 box of refillable tips (2 ul-200 ul)