Developing A Protein Purification Protocol

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Developing A Protein Purification Protocol

• Billie Parker 6-14-02

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Overview

• Introduction to Chromatography
• Extraction of Protein from Cells
• Introduction to Green Fluorescent Protein
• Results
• Conclusions

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Introduction to Chromatography

• Chromatography is used to purify complex
  mixtures.
• We used a resin in a column.
• The resin binds to certain proteins. This
  purifies these proteins away from other proteins
  that do not bind to the resin.
• After the other proteins have passed through, the
  bound proteins can be released.
• There are different resins for different
  substances you want to purify.

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Hydrophobic Interaction

• The resin binds to water-hating proteins.
• Increased salt concentrations promote binding.
• The bound protein can be released by lowering the
  salt.
• We tried three different resins to see which one
  worked the best.

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Column Information

• The resin is packed into the column already.
• The substance to be purified passes through the
  resin and binds to it.
• Most molecules do not bind to the resin.

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FPLC System Components

• The buffers carry the sample through the system.
• There are two pumps to move the buffers.
• Sample is injected into the valve.
• Then the sample passes to the column.
• The UV light detector measures the amount of
  protein based on absorbance.
• The sample is divided into fractions by the
  fraction collector.

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FPLC System
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Extraction of Protein from Cells

• Getting protein out of cells is the first step in
  purification.
• We centrifuged the bacteria to get them out of
  the growth medium.
• We used two different methods to break open the
  bacteria.
• We resuspended the cells in detergent.
• We resuspended the cells and froze them.

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Centrifuge
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Extraction by Detergent

• After lysing the cells in detergent, we
  centrifuged the cells at 10,000 xg for 10 minutes
  and kept the supernatant.
• We used a particular resin that binds to only
  detergent to purify the detergent from the
  supernatant.
• We put ammonium phosphate in the extract to
  increase the amount of salt to promote binding to
  the resin.
• Then, we loaded the extract on the column.

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Extraction by Freezing

• We used TE Buffer to resuspend the cells.
• We put the cultures into the freezer at -70o for
  20 minutes.
• We let the cells thaw.
• Then we centrifuged at 10,000 xg for 10 minutes
  and kept the supernatant.
• We put ammonium phosphate in the extract to
  increase the amount of salt to promote binding to
  the resin.
• Then, we loaded the extract on the column.

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Introduction to GFP

• GFP is Green Fluorescent Protein.
• GFP is from jellyfish.
• We used bacteria that were engineered to make
  GFP.
• GFP glows when you shine UV light on it.

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Introduction to GFP

• GFP is Green Fluorescent Protein.
• GFP is from jellyfish.
• We used bacteria engineered to make GFP.
• GFP glows when you shine UV light on it.

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Results

• The graphs will show the amount of protein
  indicated by UV absorbance.
• The salt concentration starts high to allow the
  GFP to bind then it decreases to let the GFP come
  out.
• GFP was detected by shining UV light on the
  collected fractions.

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First Column Tested

• Salt shows the salt concentration in the buffer.
• UV measures total protein.
• GFP shows fractions that glow.

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Second Column Tested

• Salt shows the salt concentration in the buffer.
• UV measures total protein.
• GFP shows fractions that glow.

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Third Column Tested

• Salt shows the salt concentration in the buffer.
• UV measures total protein.
• GFP shows fractions that glow.

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Conclusions

• The octyl column did not bind to the GFP.
• Both the phenyl and butyl columns bound the GFP
  and eluted it with low salt.
• We were unable to conclude which column worked
  the best because we cannot tell how much protein
  is in the GFP fractions.