Transcript Green Fluorescent Protein_GFP_Purification by

Green Fluorescent Protein (GFP)
Purification by Chromatography
Lab
Inoculating-Growing a cell culture
• Observe sterile techniques throughout the
experiment/lab exercise
• Add 3 mls of TE solution to vial containing
ampicillin and arabinose, respectively on Feb
20.
• Add 55 mls DI water to a 250 mls Erlenmeyer
flask-heat to boiling in a microwave.
• Add single LB tablet to the flask. Let it soak for
20 minutes. Repeat heating and swirling several
times till the entire tablet is dissolved.
Inoculating-Growing a cell culture
• Cool the LB media and add 500 μl of arabinose
and ampicillin into flask. Swirl the flask to mix the
components.
• Aliquot 2 mls of liquid media into 16 culture
tubes. Store in refrigerator till Feb 23.
• Per head use three culture tubes – inoculate a
colony from +pGLO/LB/Amp/Ara,
+pGLOLB/Amp and –pGLO/LB/Amp into three
separate tubes (on Feb 23). Shake for 30
seconds.
• Place the tubes horizontally in incubator at 32°C
till Feb 24.
Purification phases
• Bacterial Concentration and Lysis – Feb
25
- Rehydrate vial of lysozyme with 1 ml of
TE buffer
• Removing bacterial debris – Feb 27
• Protein chromatography – Mar 2
Purification phase 1
• Centrifugation – Transfer from culture
tube into microtube.
- Results in pellet of bacteria found at
bottom of tube and liquid supernatant
above the pellet.
- Pour off the liquid supernatant
- Observe the pellet with UV light
Purification phase 1
• Add 250 μl of TE buffer to pellet of
bacteria – resuspend rapidly by
pipetting up and down or vortex gently
- Keep on resuspending till no bacterial
chunks are seen
- Lysozyme – break cell wall
Purification phase 2
• Removing bacterial debris:
- Centrifugation step to separate large particles of
lysed bacteria from smaller proteins like GFP
- Supernatant will fluoresce – UV exposure
- Remove supernatant into new 2 mls microtubedone immediately
Purification phase 3
• Hydrophobic interaction
chromatography:
- Thousands of endogenous protein + GFP
separation
- GFP has several stretches of
hydrophobic regions
- GFP sticks to column
- Less hydrophobic and more
hydrophillic will elute out
Four buffers used are:
• Equilibration buffer – medium salt buffer (2 M
(NH4)2SO4) – used to equilibrate or prime the
column for binding of GFP
• Binding buffer- high salt binding buffer (4M
(NH4)2SO4) + bacterial lysate
• Wash buffer – medium salt buffer (1.3M
(NH4)2SO4 – washes weak proteins (GFP
starts penetrating into column)
• Elution buffer – low salt buffer – 10mM
TrisEDTA – wash GFP from column
Successful chromatography
• Place column gently into collection tubes. Create a paper
crutch – size of match stick and wedge between
column and collection tube – impossible for air tight
seal to form and insures column will flow
• Cap the tubes in elution steps – creates air pressure
which pushes on column bed causing sample to flow
faster
• The GFP flow out – UV light
• If column is disturbed – no elution as distinct ring –
elute out as irregular and distorted shape.
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implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60).
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