Lisa Yoneda
• Mira Mesa High School Teacher
– Biology to AP Biology
– Biotechnology
• Background:
– BS Molecular Biology
– LSSI alumni (2nd year)
– Coordinate UCSD ScienceBridge Kits
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LABORATORY 6: 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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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
10 tubes of 15 mls of Elution Buffer/ kit (EB)
Aliquot
N/A
Storage Temp
4o
N/A
N/A
RT
RT
10 tubes of 15 mls of Wash Buffer/kit (WB)
N/A
RT
10 tubes of 15 mls of Column Equilibration
Buffer/kit (CEB)
10 tubes of 5 mls of Binding Buffer/kit (BB)
N/A
RT
N/A
RT
10 tubes of 15 mls of Ethanol/kit (EtOH)
N/A
RT
1.6 mls of Lysis Buff/class (LyB)
150ul/group
4o
Equipment/Supplies
10 Student boxes with the following:
1 p20 micropipette
1 p200 micropipette
1 p1000 micropipette
1 waste and
1 ice bucket
4 Mini centrifuges
1 Tabletop centrifuge
1 Water bath
10 Ring stands with clamps
10 Boxes of p1000 tips
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Notes
Please return flask
Please return columns
50 mls extra/kit
Please return ALL tubes
50 mls extra/kit
Please return ALL tubes
50 mls extra/kit
Please return ALL tubes
20 mls extra/kit
Please return ALL tubes
50 mls extra/kit
Please return ALL tubes
1 microfuge rack
1 bag of microfuge tubes
1 bag of microfuge tubes
1 box of refillable tips (2 ul-200 ul)
Warm Up
• Why is your transformed bacteria red?
– What did you do?
– What did the bacteria do?
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Transformation
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Transcription & Translation
• Where is rfp?
• Is rfp the only
protein made by
the cell?
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Why purify a protein?
• The protein is in living cells and mixed in with
other proteins, over 1, 000 proteins could be
in one cell
• Pharmaceutical companies want one purified
protein to sell as a medicine.
• Don’t want other proteins interfering with the
medicine or body chemistry
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Protein Purification
Procedure
1. Centrifuge
Purpose
• increase concentration
2. Lyse cells- overnight
incubation
• Release cell proteins
(including rfp)
3. Centrifuge
• Separate large cell pieces
from released proteins
4. Column Purification
• Separate rfp from other cell
proteins
Where’s your protein?
• How will you know where your protein is?
• Each step of the procedure
– Record where rfp should be (prelab)
– As you complete- check location of rfp!
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Protein Purification
Procedure
1. Centrifuge
Where is rfp?
• Cytoplasm inside the cell
2. Lyse cells- overnight
incubation
• Liquid portion (cells cut
open)
3. Centrifuge
• Supernatant (liquid)
4. Column Purification
• Attach to resin in column
and then release
Step 1: Centrifuge
Step 2: Lyse cells
PART A (DAY 1)
Outline Protocol (prelab)
Protocol
• Be sure to note where the
rfp should be for each step
• Flow chart of each step
– Enough details that can use
their notes to do the lab
Notes/Changes
Part A (Day 1) Overview
• Step 1: Centrifuge
– 1 mL of rfp to microfuge tube
– Centrifuge at high speed for 5 min
– Remove supernatant without disturbing pellet
– Repeat in the SAME microfuge tube!
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Step 1: Centrifuge
• What will happen when you centrifuge your
cell culture?
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Step 1: Centrifuge Flow Chart
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Part A (Day 1) Overview
• Step 2: Lyse cells
– Add 150 µL EB (buffer) to cell pellet
– Re-suspend cells
• No clumps- use spiral notebook
• Increase surface area for lysozyme
– Add 150 µL LB (lysozyme) to cells and mix- use
spiral notebook
– Incubate Overnight (room temp)
• Can freeze if longer
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Step 2: Lyse Cells
• What will happen when we lyse the cells?
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Step 2: Lyse Cells Flow Chart
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Complete Part A (Day 1)
• Step 1: Centrifuge
• Step 2: Lyse cells
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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 how can
we isolate rfp?
• We used an expression vector
• The cells are making much more rfp than any
other protein
• Column chromatography:
• We need to understand the amino acid make up
of the rfp
• Hydrophobic and hydrophilic regions
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RFP Structure
• What will happen if you place a
folded protein in a high salt
solution?
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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
– Use cap if no stop cock
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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
• 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 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
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 Prep
• Setup columns
–
–
–
–
Arm/finger clamp
Ring stand
Waste container
Column tip should be just above waste
container
• Drain Storage buffer
• Add and drain 2 mL of equilibration buffer
• Note: ALWAYS leave ~2 mm of solution
above resin
– This is when you add next buffer
– Never drain resin dry
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Step 3: Centrifuge
Step 4: Column
PART B (DAY 2)
Outline Protocol (prelab)
Protocol
• Be sure to note where the
rfp should be for each step
• Flow chart of each step
– Enough details that can use
their notes to do the lab
Notes/Changes
Part B (Day 2) Jobs
• Step 3: Centrifuge (1 group member)
• Step 4: Column
– Column prep (1 group member- 1st class only)
– Run column
• Step 5: Column Storage/Prep for next class
• Timing is key! 3 group members needed
– Centrifuge, Column prep, gather supplies
– Run column (all members together)
– Prep/Store column, clean up, check results
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Part B (Day 2) Overview
• Step 3: Centrifuge (1 group member)
– Separate cytoplasm proteins from cell walls and
membranes
– 200 uL supernatant into clean tube
– Add 200 uL binding buffer (BB)
– Optional extension: Add 25 uL of bromocresal
green
• Visual demonstration of separation
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Step 3: Centrifuge
• What will happen when we centrifuge the lysed
cells?
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Step 3: Centrifuge Flowchart
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Part B (Day 2) Overview
• Step 4: Column
– Column prep (1 group member- 1st class only)
• Drain storage buffer
• Run Column Equilibration Buffer (CEB)
– Run column
• Binding Buffer (BB)
• Wash Buffer (WB)
• Elution Buffer (EB)
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Step 4: Run Column
• Where is rfp for each step?
– Run column
• Binding Buffer (BB)
• Wash Buffer (WB)
• Elution Buffer (EB)
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Step 4: Run Column Flowchart
• Always keep ~ 2 mm of solution above resin
– then add next solution
• Only collect red liquid- let clear flow into waste
• Add buffers slowly down sides- no clouds
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Optional Extension
• Do rfp and bromocresal green come out of the
column at the same time?
– Why or why not?
– Which one is more hydrophobic? How do you
know?
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Step 5: Column Storage
• Storage
– Run 2 mL of Storage Buffer (20% ETOH)
– Add 1 mL of storage buffer to top of resin
– Replace top & bottom caps
• Top cap should snap on tightly
• Another class
– Run 2 mL of column equilibration buffer (CEB)
– Replace top & bottom caps
• Top cap should snap on tightly
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Part A (Day 1) Teacher Prep Notes
• Can pre aliquot 1000 ul rfp into student tubes
(1/group)
– Have students pre-label tubes
– Some down time- can have previous class aliquot
next class solution
– 1st to class gets to aliquot
• Pre-label collection rack (1/period)
• If have sensitive centrifuge recommend having
students use a balance for 2nd spin
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Part B (Day 2) Teacher Prep Notes
• Columns run slow
– Can prep 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
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Applying Pressure
• Can make plunger to increase column flow rate
– #2 stopper- 1 hole
– 10 mL syringe
– Attach together by cutting 1 mL disposable pipet
• Using plunger
– 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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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
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