From a Cell to a Gel:

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From a Cell to a Gel

• In Search of Proteins!

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From the Cell
To the Gel
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(No Transcript)
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How do we study proteins?

• Make recombinant proteins
• Baculovirus an insect cell virus, particularly
  useful for study of eukaryotic proteins
• E.coli bacteria generally used, especially for
  less complex protein
• Purify proteins for functional analysis

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Baculovirus Lifecycle
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Making proteins with baculovirus

gene
Baculovirus DNA
Recombinant protein
insect cell
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Insect cells
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Identifying proteins the gel...
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Workstation 1
Workstation 2
Insect cells under the microscope
Sample pipetting
Workstation 3
Workstation 4
Analysis of electrophoresis
Gel loading
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BioRad Kit Protocol

• Transform bacteria with pGLO plasmid
• Select for ampicillin resistance
• Induce expression of GFP with arabinose
• Grow transformed colony and prepare lysate
• Purify GFP using column chromatography

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What is a plasmid?

• Circular piece of autonomously replicating DNA

• Originally evolved by bacteria
• May express antibiotic resistance
• or be modified to express proteins of interest

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pGLO Plasmid

• Beta Lactamase
• Ampicillin resistance
• Green Fluorescent Protein
• Aequorea victoria jellyfish gene
• araC regulator protein
• Regulates GFP transcription

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What is transformation?

• Uptake of foreign DNA often a circular plasmid

GFP
Amp Resistance
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pGLO Transformation
Cell wall
GFP
Bacterial chromosomal DNA
Beta lactamase (ampicillin resistance)
pGLO plasmids
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pGLO Regulation
ara Operon
araC
B
A
D
Effector (Arabinose)
Effector (Arabinose)
araC
B
A
D
RNA Polymerase
B
A
D
araC
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Transformation Procedure

• Suspend colonies in Transformation Solution
• Add loop of pGLO plasmid DNA to tube
• Incubate tubes 10 minutes on ice
• Heat shock 50 seconds at 42oC
• Incubate 2 minutes on ice
• Incubate 10 minutes with LB broth
• Plate

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Why perform each transformation step?

• 1. Incubation on ice slows fluid cell membranes
• 2. Heat-shock increases permeability of cell
  membrane
• 3. LB recovery allows beta lactamase expression

Cell wall
GFP
Beta lactamase (ampicillin resistance)
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DPTP Volume Measurement
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Steps in GFP Purification

• Grow bacterial culture expressing GFP
• Pellet the bacteria by centrifugation

spin to pellet
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Purification Steps continued

• Lyse the bacteria by freeze/thaw and lysozyme
  treatment

• Centrifuge to pellet the cellular debris

• Load supernatant

spin to pellet
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Load gels and perform electrophoresis