Lab 6

1
Lab 6

• Chloroplast extraction
• TLC Column Chromatography

2
Order of events for Exercise 6

• Divide each group into two sections. One group
  will do the organic separation protocol and one
  will do the aqueous separation protocol.
• Since students are responsible for both
  procedures, they need to share the experience and
  findings.
• All work done with organic solvents must be done
  in the hood. Work with aqueous solutions may be
  done at the students workstation.

3
(6.1-6.5) Isolation of chloroplasts and
extraction and separation of pigments with TLC

• Selecting spinach leaves
• Placing cells in a protective environment
  (chloroplast isolation medium)
• Breaking open cells (blender)
• Separating organelles (chloroplasts) of interest,
  filtering centrifugation
• Homogenizing chloroplasts
• Disrupting membranes
• Separation of pigments with TLC
• Spectral analysis of leaf pigments

4
1. Selecting an Appropriate Source of Cells
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2. Placing Intact Cells in a Protected Environment

• Osmotic support
• pH and ionic composition
• Reducing agents
• Chelators

6
REMOVE GLASS ROD!
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Low Temperature

• Breaking Open the Cells
• Separating the Organelle of Interest from Other
  Cell Components

8
BALANCE TUBES OF EXTRACTS
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Differential centrifugationRCF 200 X g --gt 1100
X g
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Organic extraction of lipid-soluble pigments
Chloroform layer
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Developed Chromatogram

• Chromatogram removed from chamber when fastest
  moving lipid nears top end of the sheet.
• Separated lipids could be removed from adsorbent
  sheet and dissolved in liquid.

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Measuring Rf
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TLC
A B-carotene B chlorophyll a C chlorophyll
b D,E,F xanthophylls
http//www.msu.edu/course/lbs/145/luckie/inquiries
2006/vitaC/index.html
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CHLOROPHYLL A 400-450 nm 650-700 nm CHLOROPHYLL
B 450-500 nm 640 nm
http//en.wikivisual.com/images/7/73/Chlorophyll_a
b_spectra.png
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(6.5-6.7) Separation of water soluble-proteins by
Ion-Exchange column chromatography

• Adding initial 0.005M Tris to DEAE-cellulose
• Adding phycoerythrin phycocyanin extract
  mixture to top of column
• Allowing extract to become embedded in
  DEAE-cellulose bed
• Preparing collecting cuvettes
• Separating pigments with the addition of
  increasing concentrations of Tris
• (0.005M, 0.01M, 0.05M, 0.10M, 0.25M, 0.5M)
• Spectral analysis of purified aqueous pigments

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Ion Exchange Column Chromatography
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Dislodging Protein Molecules

• After negatively charged proteins have adhered to
  the DEAE-cellulose, salt solution of different
  concentrations are used to displace the proteins.
• Weakly adhering protein molecules can be
  dislodged with dilute salt solutions that have
  (-)ions with stronger negative charges.
• Stronger adhering proteins can be dislodged with
  more concentrated salt solutions.

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SAFETY

• EVERYONE WEARS A LAB APRON GLOVES TODAY
• Organic solvents
• Carcinogenic and toxic
• Use in hood only
• Use gloves, lab apron, and goggles
• Do not break the glass tube for homogenizing and
  do not grip warms tube up
• Do not leave glass rod in blender
• Follow all other safety guidelines not listed here

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DISPOSAL

• All samples containing anything organic are to be
  disposed in proper containers in the hood
  organic liquid waste, solid waste, and glass
  waste
• All DEAE columns are recycled by prep staff do
  not do anything with them at the end
• Return chloroplast isolation media to fridge
• Cheesecloth etc in solid chemical waste