Lisa Yoneda Mira Mesa High School Teacher – Biology to AP Biology – Biotechnology Background:...

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www.amgenbiotechexperience.com

Lisa Yoneda

• Mira Mesa High School Teacher– Biology to AP Biology– Biotechnology

• Background:– BS Molecular Biology– LSSI alumni (2nd year)– Coordinate UCSD ScienceBridge Kits

LABORATORY 6: GETTING WHAT WE NEED-PROTEIN

LSSI AlumLisa Yoneda, Biotechnology Program, Mira Mesa HS

SafetyGeneral 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

Lab Prep & Aliqouting GuidelinesReagents/Supplies Aliquot Storage Temp Notes1 flask of 250 mL of LB/Amp/Ara broth (OR use plate from Lab 5 to inoculate/Kit

N/A 4o Please return flask

10 Resin packed columns N/A RT Please return columns10 tubes of 15 mls of Elution Buffer/ kit (EB) N/A RT 50 mls extra/kit

Please return ALL tubes10 tubes of 15 mls of Wash Buffer/kit (WB) N/A RT 50 mls extra/kit

Please return ALL tubes10 tubes of 15 mls of Column Equilibration Buffer/kit (CEB)

N/A RT 50 mls extra/kitPlease return ALL tubes

10 tubes of 5 mls of Binding Buffer/kit (BB) N/A RT 20 mls extra/kitPlease return ALL tubes

10 tubes of 15 mls of Ethanol/kit (EtOH) N/A RT 50 mls extra/kitPlease return ALL tubes

1.6 mls of Lysis Buff/class (LyB) 150ul/group 4o

Equipment/Supplies10 Student boxes with the following:

1 p20 micropipette 1 microfuge rack1 p200 micropipette 1 bag of microfuge tubes 1 p1000 micropipette 1 bag of microfuge tubes1 waste and 1 box of refillable tips (2 ul-200 ul)

1 ice bucket4 Mini centrifuges1 Tabletop centrifuge1 Water bath10 Ring stands with clamps10 Boxes of p1000 tips

Warm Up

• Why is your transformed bacteria red?– What did you do?– What did the bacteria do?

Transformation

Transcription & Translation

• Where is rfp?

• Is rfp the only protein made by the cell?

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

Protein Purification

Procedure Purpose1. Centrifuge

2. Lyse cells- overnight incubation

3. Centrifuge

4. Column Purification

• increase concentration

• Release cell proteins (including rfp)

• Separate large cell pieces from released proteins

• 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!

Protein Purification

Procedure Where is rfp?1. Centrifuge

2. Lyse cells- overnight incubation

3. Centrifuge

4. Column Purification

• Cytoplasm inside the cell

• Liquid portion (cells cut open)

• Supernatant (liquid)

• Attach to resin in column and then release

PART A (DAY 1)

Step 1: Centrifuge

Step 2: Lyse cells

Outline Protocol (prelab)

Protocol Notes/Changes• 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

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!

Step 1: Centrifuge• What will happen when you centrifuge your cell

culture?

Step 1: Centrifuge Flow Chart

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

Step 2: Lyse Cells

• What will happen when we lyse the cells?

Step 2: Lyse Cells Flow Chart

Complete Part A (Day 1)

• Step 1: Centrifuge

• Step 2: Lyse cells

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?

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)

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

RFP Structure

• What will happen if you place a folded protein in a high salt solution?

Column Structure

• Column• Stop Cock• Matrix• Solvents

Column Structure

• Column– Glass (or plastic) tube that holds

matrix• Stop Cock– Regulates flow of solvents• In line: allows solvents to flow• Perpendicular: blocks solvents-

stops flow

– Use cap if no stop cock

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

Column Structure

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 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

Wash Buffer

• Medium Salt Buffer– 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

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 =

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

PART B (DAY 2)

Step 3: CentrifugeStep 4: Column

Outline Protocol (prelab)

Protocol Notes/Changes• 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

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

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

Step 3: Centrifuge

• What will happen when we centrifuge the lysed cells?

Step 3: Centrifuge Flowchart

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)

Step 4: Run Column

• Where is rfp for each step?– Run column• Binding Buffer (BB)• Wash Buffer (WB)• Elution Buffer (EB)

• 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

Step 4: Run Column Flowchart

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

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

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

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

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

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

Lisa Yoneda Mira Mesa High School Teacher – Biology to AP Biology – Biotechnology Background:...

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