Encyclopedia of Experiments: Biological Techniques
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Transcript
Fourier transform infrared spectroscopy — FTIR — helps to study peptide self-assembly into supramolecular structures held together by non-covalent interactions.
Begin by taking an unassembled protein sample — a suspension of amyloid peptides with the ability to form a beta sheet-rich supramolecular structure. Place the sample in an FTIR instrument equipped with an ATR crystal.
Direct an infrared beam at a critical angle onto the crystal; the beam passes through it and hits the interface between the crystal and sample. Crystals with a higher refractive index than the sample cause internal reflection of the beam.
A small fraction of the beam extends into the sample as an evanescent wave — a standing wave at the reflection points. Interaction of this wave with the sample causes absorption of the wave's energy, leading to attenuation of the reflected beam, recorded by a detector.
Depending on the peptide's structure, the sample absorbs in specific regions of the infrared spectrum. The absorption spectrum — obtained by Fourier transformation of the signal — shows a broad peak at a specific wavenumber, characteristic of the unassembled protein sample.
Incubate the sample, allowing self-assembly, to form beta sheet-rich amyloid fibers. Post-incubation, record the absorption spectrum.
A sharp peak at a wavenumber different from the previous broad peak indicates conversion into a beta-sheet assembly.
