Isolation and Spectral Characterization of Chlorophyll-Protein Complexes
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Isolation and Spectral Characterization of Chlorophyll-Protein Complexes of Chloroplast Thylakoid Membranes
Introduction

In the undergraduate biology laboratory, labs on photosynthesis ordinarily fall into a small
number of categories:
1. measurement of the overall process of photosynthesis by carbon dioxide uptake or
oxygen evolution
2. measurement of the Hill reaction (the light driven reduction of an exogenous dye with
electrons from water) in isolated chloroplasts
3. pigment analysis by thin-layer or paper chromatography
Our overall goal is to introduce modern molecular techniques into the teaching of plant science
at IUP. We are doing this at three levels in three courses:
• Introductory Botany (BIOL 210), a requirement for all biology majors (and usually taken by
sophomores), is part of the Diversity Core of the Biology curriculum. It is an elective for
biochemistry majors. The “green gel” lab presented here is part of this course. Additionally,
we use PCR and Koch’s Postulates to identify Agrobacterium as the causative agent of
sunflower tumors.
• Plant Physiology (BIOL 350/550) is an elective for both biology and biochemistry majors.
We use a reporter gene to investigate gene expression, and plant tissue culture to transform a
reporter gene into plant tissue.
• Laboratory Methods (BIOL 499) is a requirement for biochemistry majors and an elective for
biology majors. Here we are actually introducing plant science to the lab methods
curriculum. We perform in vitro translation of a plant virus and analyze it using Western
Blots and perform a physical and restriction analysis of an amplified target gene.
Our Botany course has no distinction between lecture periods and lab periods. This lab is
performed as part of the unit on photosynthesis. It is performed by groups of eight students (two
groups of four) with the instructor demonstrating and “cheering them on”. We use Spinach as the
prime example that we know will work, and plants grown at high and low light intensities to show
the plasticity of the photosynthetic apparatus.
Prior to the lab, the instructor sets up the gel apparatus with gels. The lab is set up in a flow
pattern with stations for each procedure since we have only one refrigerated centrifuge and
Chlorophyll-Protein Complexes 133
spectrophotometer. The electrophoresis and cutting out gel bands is done at the students’ tables in
groups of four.
At the beginning of the lab period the instructor briefly reminds the students of relevant lecture
material, outlines the procedures, and gives safety instructions. With everyone looking on, the
instructor demonstrates the procedures first and then allows the students to take over.
After the electrophoresis is completed, the instructor selects one group’s gel and places it on an
overhead projector for discussion and comparison. The lab continues with the measurement of
absorption spectra of gel bands. The spectra are discussed during a later class period.
In our Botany course this exercise, as described here, is the goal. More advanced classes can use
this as the starting point. Examination of the electrophoretic pattern and absorbance spectra of
leaves subjected to various environmental perturbations can make a multi-week project. Advanced
biochemistry labs can subject each band to polypeptide analysis using the LaemmLi system.
Further, the Photosystem I complexes contain an active P700 which can be detected by oxidizedminus-
reduced or light-minus-dark difference spectra.

Download full report
http://ablewebvolumes/vol-24/9-dietrich.pdf
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