BIOL2200 Lecture Notes - Lecture 7: Polyclonal Antibodies, Primary And Secondary Antibodies, Vimentin
BIOL2200 L1 Wk 3
immuno
Antibodies
histo
Fixed tissue/sectioned tissue
Visualising where a protein is expressed
Organ -> Cell type -> Location within the cell
Eg, Heart -> cardiomyocyte -> nucleus
visualising where proteins are localised within a cell using IHC/IF
Ball of cells, not much detail,
apical protein, forms a lumin, can see that there are 6 cells, better picture of whats going on
Labelling organelles within a cell
If youre trying to figure out if your gene is involved, then you can quantify the mitochondria and ask
'under the mutany situation, how does this change'
Quantifying synapses in neurons
Purple is the motor neuron, the active zones/synapses of the muscle
1. Fewer synapses
2. More synapses in them, and also bigger motor neurons
An antibody contains variable and constant domains
Antibodies contain immunoglobulin (Ig) protein fold that are also found in other proteins
How do you make an antibody?
How do you know that your antibody binds to the antigen you think it does?
oWhat are essential controls?
Polyclonal vs monoclonal antibodies
1. Polyclonal - many species
Antibodies to multiple antigenic epitopes contained within serum
Antibodies against the same protein, but antibodies bind to different antigens on the
cell, polyclonal antibodies will contain antibodies from multiple types of b cells
More antibodies that can bind to protein, better reactivity
1. Monoclonal-mouse and rat only (some rabbits)
Antibody recognises a single antigenic epitope
oAntibodies from the same b cell
oClone the cell that produces the antibody, immortalise it so you can produce it
oImportant for more specific tools, raise an antibody against a phosphorylation site
eg. Can recognise when this particular amino acid was phosphorylated, but not when it
wasn't
Using antibodies to detect proteins
1. Unconjugated antibody
1. Conjugated antibody
IHC procedure
Using multiple antibodies in the same tissue
IHC in prac 1
Youll be using conjugated primary antibody against a protein called vimentin
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Organ -> cell type -> location within the cell. Eg, heart -> cardiomyocyte -> nucleus visualising where proteins are localised within a cell using ihc/if. Ball of cells, not much detail, apical protein, forms a lumin, can see that there are 6 cells, better picture of whats going on. If youre trying to figure out if your gene is involved, then you can quantify the mitochondria and ask. "under the mutany situation, how does this change" Purple is the motor neuron, the active zones/synapses of the muscle. More synapses in them, and also bigger motor neurons. Antibodies contain immunoglobulin (ig) protein fold that are also found in other proteins. Antibodies to multiple antigenic epitopes contained within serum. Antibodies against the same protein, but antibodies bind to different antigens on the cell, polyclonal antibodies will contain antibodies from multiple types of b cells. More antibodies that can bind to protein, better reactivity.
