Lec7 Flashcards
Immunofluorescence
Antibodies covalently bonded to fluorescent dye
Antibodies have an affinity for a target and will glow when attached to the target
IgG
Antibody
How to make an antibody
Inject a protein into an animal
Wait for animal to make antibodies against it
Purify all antibodies and isolate the ones that stick to the target
Takes a long time to do
YFP
Your fav protein
Direct immunofluorescence
Target sticks to antibody which is attached to dye
Indirect immunofluorescence
Primary antibodies are attached to the target
Secondary antibodies attach to primary and have the dye
More steps but also more fluorescence
Fluorescent fusion proteins
Fluorescent proteins are made of GFP (green fluorescent protein)
By making modifications to the gene, it will have different fluorescence
Fluorescent fusion proteins
Method
Make a cell that is one part YFP and one part GFP
To make this you need a mRNA that binds to both
To make the mRNA you need a plasmid with YFP attached to the GFP
Microtubule Fluorescent fusion proteins example
Fusion protein is attached alongside regular microtubules by the side with the microtubule on it rather than the dye. The dye allows the presence of the microtubule to fluoresce
Nomenclature for fusion protein
First protein (colon) second protein
Tagged fusion protein
YFP is attached to HA-tag which is made by plasmids
Fusion proteins can be detected through anitbodies rather than dye
Mix of the other two methods
Methods to eliminate specific proteins of the cell
Can act at the DNA RNA or protein level
DNA level
Gene mutation
Cells with a nun functioning gene cannot make YFPs
Delta YFG
Deletion of your favorite gene
Morpholinos
RNA level
Synthetic mRNA molecules that bind to target mRNAs that block ribosomes
