Lecture 2 Flashcards
T/F: Antibodies are produced by all mammals
True
Structure of the antibody?
-four individual polypeptide chains
-Botoom of the Y is constant in species
-Antigen biding sites are identical but are different in every antibody
Epitopes?
Parts of the antigen that bind to the antigen binding sites
T/F: Antibodies are made against full proteins?
False, typically only against a part of a protein
What is the disadvantage of immunisation for creating antibodies(polyclonal)?
Polyclonal: Multiple antibodies that have the same affinity for the same antigen but different epitopes. Antibodies might not bind to the regions of the protein we want
T/F: The more various polyclonal antibodies in a mixture the higher the risk of off targeting?
True, more polyclonal antibodies = less specific staining
Another problem with immunisation for production of antibodies?
Some proteins produce a good immune response but proteins too similar to ones already in the animal do not
Monoclonal Antibodies
All of the antibodies are identical and have the same constant/variable regions bind the same epitopes
How are antibodies made?
B cells produce them and make the same antibody for life.
Do B cells express telomeres?
No they cannot divide forever
Hybridoma?
Hubrid of the B cell that can produce identical antibodies and divide forever
Problem with hybridoma production?
-B-cells, cancer cells and hybridoma cells all end up in one container
-The cancer cells outcompete the hybridomas
How do we get rid of B-cells?
B cells are primary cells (moralized). To get rid of them we allow them to grow/divide until they reach a crisis
How do we get rid of cancer cells?
Use cancer cells that lack an enzyme that allows them to survive in a media called HAT
Hybridomas survive in HAT and cancer cells die
How to determine each well only contain one hybridoma?
Cut the target protein into pieces and place them into the well. If the hybridomas only bind to one piece we can conclude there is only one hybridoma type
Advantages of hybridomas?
-Immortal
-Monoclonal( highly specific staining)
Microinjection
-A way to get antibodies into the cell
-Stick a needle containing the antibody into the cell
Does immunofluorescence work in live cells?
No
Advantages of indirect immunofluorescence?
-Multiple fluorescent antibodies can bind the primary antibody to create twice as much fluorescence
WHat type of fix is used in immunofluorescence?
Formaldehyde or methanol
Glutaraldehyde can also be used it is strong only need a little but it does have fluorescence could cause background noise
What do we preameabilize cells with ?
Methanol
When do we not have to fix a cell in immunofluorescence?
If the protein of interest is on the outside of the cell
T/F: tissues are normally sectioned prior to staining?
True
Multiple fluorescent labels
Using multiple antibodies to view various proteins
Indirect immunofluorescence with multiple fluorescent labels?
-Each color must be from a different animal
-If two are from the same animal the secondary antibodies would both bind to both primary types
Immunogold
Attaching gold instead of fluorescence to the end of an antibody and then doing EM
GFP fusion protein
Gene for GFP is attached to the gene encoding for protein of interest
T/F: GFP fusion protein can be used to localize proteins?
Yes
Time delay for GFP fusion?
Takes up to 24 hours for the cell to start expressing the GFP fusion protein after transfection
Advantages of GFP
-Used in both living and fixed cells
-Live imaging
-No antibodies
-Can use other colours
Does the GFP protein have to be stuck onto the C-terminal ?
No N-terminal or C-terminal is fine
Disadvantages of GFP in terms of folding?
Sometimes GFP fusion proteint do not fold properly (dont behave the same way as real protein)
Disavantage of GFP fusion protein amount?
Protein being expressed may not be expressed at the same rate in a physiological cell
-Proteins do different things at different concentrations
Disadvantage of GFP fusion endogenous?
The cell now has two copies of the same protein just now one is GFP labelled
One might have priority over the other could affect the others function
