Fluorescent Proteins Flashcards
What is a major characteristic of fluorescent dyes and what is the active group in fluorescein?
multiple rings
Isothiocyanate enables binding to primary amines.
How are secondary antibodies often modified?
Attach an enzyme e.g. alkaline phosphatase
Pi produced in presence of certain chemicals –> coloured ppt.
What is phalloidin?
toxin from death cap fungus
Fluorophores (such as rhodamine) ADDED to OH groups
Binds and stabilises F ACTIN but kills cells at high concentrations–> only used fixed cells but no need for antibodies.
Define;
Quantum yield
Stokes shift
Lifetime
- ratio of emitted photons to absorbed photons
- difference in wavelengths
- time taken to emit a photon.
What experiments have been carried out with fusion proteins?
Comparison between fibroblasts from wild type and integrin null mice.
Transgenic drosophila transfected with H2B-GFP showed development of embryo nuclei.
What molecules can be used in FRET?
fluorescent proteins
fluorophore labelled antibodies.
what are the different types of fluorescence and their uses?
Others (antibodies or fluorescent dyes used to track localisation of cellular proteins).
Fusions used to follow cells during development.
FRET - used to study when and where two proteins interact.
What are the first two steps hat just be carried out when using antibodies?
Inject protein into animal to create antibody
Purify antibody using antigen.
(Then stain using protein specific antibody).
