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Developing A Protein Purification Protocol. Billie Parker 6-14-02. Overview. Introduction to Chromatography Extraction of Protein from Cells Introduction to Green Fluorescent Protein Results Conclusions. Introduction to Chromatography. Chromatography is used to purify complex mixtures.

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Presentation Transcript
overview
Overview
  • Introduction to Chromatography
  • Extraction of Protein from Cells
  • Introduction to Green Fluorescent Protein
  • Results
  • Conclusions
introduction to chromatography
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.
hydrophobic interaction
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.
column information
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.
fplc system components
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.
extraction of protein from cells
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.
extraction by detergent
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.
extraction by freezing
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.
introduction to gfp
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.
introduction to gfp13
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.
results
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.
first column tested
First Column Tested
  • Salt shows the salt concentration in the buffer.
  • UV measures total protein.
  • GFP shows fractions that glow.
second column tested
Second Column Tested
  • Salt shows the salt concentration in the buffer.
  • UV measures total protein.
  • GFP shows fractions that glow.
third column tested
Third Column Tested
  • Salt shows the salt concentration in the buffer.
  • UV measures total protein.
  • GFP shows fractions that glow.
conclusions
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.