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Botany JoVE (Open Access) Citable · DOI

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

DOI: 10.3791/51852-v
What you'll learn
  • ✓ Extract and purify pigments from plant tissue in vitro
  • ✓ Reconstitute light-harvesting complexes with extracted chlorophyll and carotenoids
  • ✓ Characterize reconstituted LHCs using chromatography and centrifugation techniques
  • ✓ Validate functional properties of reconstituted complexes against native standards
Protocol

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

Difficulty
advanced
Total time
~6–8 hours per batch (pigment extraction through final characterization)
Model organism
Spinacia oleracea (spinach)

Steps

1
Extract total pigments from spinach leaves

Harvest and process fresh spinach leaf tissue to extract chlorophylls and carotenoids. Isolate the soluble pigment pool required for subsequent complex reconstitution.

▶ 01:24
2
Reconstitute light-harvesting complexes in vitro

Mix purified apo-proteins with extracted pigments under controlled conditions to allow spontaneous assembly of functional light-harvesting complexes that coordinate chlorophylls and carotenoids.

▶ 04:52
3
Purify complexes using nickel column chromatography

Apply reconstituted complexes to immobilized metal affinity chromatography to isolate and purify his-tagged light-harvesting complex proteins.

▶ 07:37
4
Separate complexes by sucrose gradient centrifugation

Subject purified samples to density-based ultracentrifugation on sucrose gradients to achieve further separation and characterization of reconstituted LHC populations.

▶ 08:18
5
Validate properties of reconstituted light-harvesting complexes

Analyze optical and biophysical properties of reconstituted complexes to confirm they match native complex behavior, including pigment composition and light-absorption characteristics.

▶ 09:21
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