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 microtube-done 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.

• This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis:

• against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and

• against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

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