Single cell methods Flashcards
What is immunohistochemistry? Is live imaging possible?
Methods of detecting and localizing proteins using antibody labeling. Uses and antibody (that is coupled to a fluorophore or an enzyme) that binds to the protein of interest.
Live imaging is not possible with this method, since the cell structures need to be fixed with formaldehyde.
What is a fluorescent reporter and can it be used for live imaging?
A fluorescent gene fused to the GOI - when the promoter/gene is expressed then the reporter protein absorbs a light at one wavelength and emits another wavelength - visible.
Live imaging is possible with this method.
What is the difference between a transcriptional and translational reporter?
Trancriptional reporter - the reporter gene is not translationally fused to the protein - may only be expressed when the promoter is active.
Translational reporter - reporter gene is translationally fused to the sequence of the gene of interest = reporter protein and protein of interest will be fused. Can be used to detect sub cellular location of protein.
What is an epitope tag and what is it used for?
An epitope is a sequence translationally fused to a gene of interest. When the gene is translated, an additional protein will be fused to the POI. This can be used as an antigen to which antibodies can bind.
What is FISH (also smFISH) and what is it used for?
Fluorescent in situ hybridization methods. Methods used to detect tissue specific and subcellular localization of DNA and RNA (mainly RNA transcripts). smFISH stands for small molecule FISH and is based on the base pairing of small, fluorescently labeled RNAs to target molecules.
How does FISH work and how is the detection of the fluorescence performed?
