Transcript pGLO™ GFP Purification — Electrophoresis and - Bio-Rad

What is next after pGLO™ bacterial transformation?
Instructors
Stan Hitomi
Coordinator – Math & Science
Principal – Alamo School
San Ramon Valley Unified School District
Danville, CA
Kirk Brown
Lead Instructor, Edward Teller Education Center
Science Chair, Tracy High School
and Delta College, Tracy, CA
Bio-Rad Curriculum and Training Specialists:
Sherri Andrews, Ph.D.
[email protected]
Essy Levy, M.Sc.
[email protected]
Leigh Brown, M.A.
[email protected]
Why Teach
Bacterial
Transformation
and Protein
Purification?
• Powerful teaching tool
• Laboratory extensions
• Real-world connections
• Link to careers and industry
• Standards based
Workshop
Time Line
• Introduction
• Background on GFP
• Protein Electrophoresis of GFP
• Purify GFP using column chromatography
Starting Point:
Transformation
Procedure
Overview
Day 1
Day 2
…
Discovery of GFP
• Originally Isolated from the jellyfish
Aequorea victoria
• Naturally occurring in many
bioluminescent jelly fish, reef corals
and marine crustaceans
• Recombinant GFP has 239 amino acids
• Expressed as a 26,870 Dalton protein
• Barrel structure with the fluorescent
chromophore at center of the protein
• “Nobel prize-winning” molecule!
What is a
chromophore?
A group of atoms
and electrons
forming part of an
organic molecule
that causes it to
be colored
The GFP
chromophore is
comprised of
three adjacent
amino acids.
These amino
acids are
enzymatically
converted to an
active cyclic
chromophore
GFP
Chromophore
Absorbs at 395 nm
Emits at 509 nm
•In vivo, GFP complexes with aequorin, a calciumbinding protein which transfers energy, to excite
GFP
•In vitro, UV light is used to excite the GFP
chromophore, absorbing light at a wavelength of
395 nm, and emitting at a longer wavelength of
509 nm visible fluorescent green light
Recombinant
GFP
GFP has been mutated
• Increased fluorescent photostability
• Improved hydrophilicity
• Increased solubility
• Improved fluorescence
• Various colors available
• Improved use as a reporter protein
Using GFP as a biological
tracer or reporter protein
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html
With permission from Marc Zimmer
Links to
Real-world
• GFP is a visual marker
• Study of biological processes
(example: synthesis of proteins)
• Localization and regulation of gene
expression
• Cell movement
• Cell fate during development
• Formation of different organs
• Screenable marker to identify transgenic
organisms
www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
Protein
Electrophoresis
of GFP
Procedures
Overview
Students will
learn:
Protein
Electrophoresis
of GFP
• Prepare an SDS-PAGE sample and understand the
components of Laemmli buffer
• Understand protein structure and mechanisms for
protein folding and unfolding and how different
conformations can be identified using
electrophoresis
• Understand how proteins are separated during gel
electrophoresis
• Understand the use of electrophoresis in the
process of transformation to protein expression
• Understand chromophores and the basis of
protein fluorescence
• Construct a standard curve and determine the
molecular weight (MW) of an unknown protein
Sample
Preparation:
SDSPolyacrylamide Gel
Electrophoresis
(SDS-PAGE)
“no heat” samples
Labeled tubes:
1. White, no heat
2. Green, no heat
3. White, +heat
4. Green, +heat
Samples should be a
“healthy scoop” of
colonies
• Label four screw-capped microtubes
• Add 300 µl of Sample buffer to the two “no heat’
tubes
• Using the inoculation loop, scrape a sample (20-100
colonies) from an LB/amp (white colonies) plate and
transfer to the “White, no heat” tube and mix
thoroughly
• Using the inoculation loop, scrape a sample (20-100
colonies) from an LB/amp/ara (green colonies) plate
and transfer to the “Green, no heat” tube and mix
thoroughly
Sample
Preparation
SDS-PAGE
“heat” samples
• Transfer 150 µl of the “White, no heat” mixture to
the “White,+heat” tube
• Transfer 150 µl of the “Green, no heat” mixture to
the “Green,+heat tube”
• Heat the “+heat” tubes to 95°C for 5 min in a
water bath. Cool to room temperature
Heating the samples
denatures the proteins
No heating
intact chromophore
Heating
denatured chromophore
functional protein
non-functional protein
fluorescent protein
non-fluorescent protein
There is an important link between the
STRUCTURE and FUNCTION of the protein
• Tris buffer
– provides appropriate pH
CH3
CH2
• SDS (sodium dodecyl sulfate)
– Solubilizes and denatures proteins
– Adds a negative charge to the
protein
CH2
CH2
CH2
• DTT
– (1,4-Dithiothreitol) reduces disulfide
bonds, to help unfold proteins and
protein complexes
CH2
CH2
CH2
CH2
• Glycerol
– Increases the density of the
samples to help samples sink into
wells of the gel
CH2
CH2
S
O
O
O
• Bromophenol Blue
– dye to visualize samples
CH2
O
What is in the
Laemmli
sample buffer?
-
SDS
Load and
electrophorese
samples
30min at 200V
in 1XTGS Buffer
UV illumination Coomassie Stain
How Does
SDS-PAGE
Work?
• Denatures proteins using detergent, DTT, and
heat
s-s
• Separates proteins based on size
• Negatively charged proteins move to positive
electrode
–
+
• Smaller proteins move faster through the gel
Why Use
Polyacrylamide
Gels to
Separate
Proteins?
• Polyacrylamide gel has a tight matrix
• Ideal for protein separation
• Smaller pore size than agarose
• Proteins much smaller than DNA
– Average amino acid = 110 daltons
– Average nucleotide pair = 649 daltons
– 1 kilobase of DNA = 650 kD
– 1 kilobase of DNA encodes 333 amino acids = 36 kD
• Size measured in kilodaltons (kD)
• Dalton = mass of hydrogen molecule
= 1.66 x 10-24 gram
• Average amino acid = 110 daltons
GFP
VisualizationDuring & Post
Electrophoresis
During Electrophoresis
Post Electrophoresis
• Fluorescent GFP can
be visualized during
electrophoresis
Fluorescent
isoform
• Coomassie stained
gels allow for
visualization of
induced GFP proteins
Nonfluorescent
isoform
Prestained bands
+ UV activated GFP
Coomassie stained
bands
Determining the
molecular
weights of GFPs
in different
conformations
GFP
Chromatography
Kit
GFP Purification
Procedures
Overview
Day 1
Day 2
Day 3
Why Use
Chromatography?
• To purify a single
recombinant protein
of interest from
over 4,000 naturally
occurring E. coli
gene products.
Column
Chromatography
• Chromatography
used for protein
purification
– Size exclusion
– Ion exchange
– Hydrophobic
interaction
Hydrophobic
Interaction
Chromatography:
(HIC)
Steps 1–3
1. Add bacterial lysate to column matrix in
high salt buffer
2. Wash less hydrophobic proteins from column in
low salt buffer
3. Elute GFP from column with
no salt buffer
Step 1:
Hydrophobic
Interaction
Chromatography
• Add bacterial lysate
to column matrix in
high salt buffer
– Hydrophobic
proteins interact
with column
– Salt ions interact
with the less
hydrophobic
proteins and H2O
Hydrophobic
bead
Step 2:
Hydrophobic
Interaction
Chromatography
• Wash less
hydrophobic from
column with low
salt buffer
– Less hydrophobic
E. coli proteins fall
from column
– GFP remains bound
to the column
O
- O S OO
Hydrophobic
bead
Step 3:
Hydrophobic
Interaction
Chromatography
• Elute GFP from
column by adding a
no-salt buffer
GFP
– Released from
column matrix
– Flows through the
column
Hydrophobic
bead
Laboratory
Quick Guide
Helpful Hints:
Hydrophobic
Interaction
Chromatography
• Add a small piece of
paper to collection tube
where column seats to
insure column flow
• Rest pipet tip on side of column
to avoid column bed disturbance
when adding solutions
• Drain until the meniscus is just
above the matrix for best separation
Column
Chromatography
vs. SDS-PAGE
for protein
isolation and
analysis
Both methods separate proteins from a
complex mixture
Column Chromatography
Used to isolate a protein
from a complex mixture of
molecules based on its
physical and/or chemical
properties
HIC separates molecules
based on hydrophobicity
Need to lyse open the
cells to run the soluble
proteins over the column
Very dilute concentration
of GFP in the HIC column
fractions
SDS PAGE
An analytical technique
used to detect the
presence of a protein of
interest
Separates molecules
based on size
Can compare denatured
and intact proteins to
study protein structure
High concentration of
GFP from whole cell
lysate samples (dense
colonies)
Both techniques, used in concert can help
scientists purify and analytically study proteins
Transformation
is only the
beginning…
pGLO Transformation
… More
techniques are
necessary to
fully understand
the structure and
nature of a
protein.
Protein Purification
Size and structure determination
Results may
lead to more
experiments!
Webinars
• Enzyme Kinetics — A Biofuels Case Study
• Real-Time PCR — What You Need To Know
and Why You Should Teach It!
• Proteins — Where DNA Takes on Form and
Function
• From plants to sequence: a six week
college biology lab course
• From singleplex to multiplex: making the
most out of your realtime experiments
explorer.bio-rad.comSupportWebinars