Microscopy II Flashcards
What are antibodies
Immunoglobulins produced by b lymphocytes and plasma cells of vertebrates
Which type of antibodies used in immunocytochemistry
IgG class of immunoglobulins
Describe antibody structures - gen
Y SHAPED
2 light chains
2 heavy chains
2 antigen biding sites, one at each tip of each arm of y
Describe antibody structure = specifics
4 polypeptide chains, makes y
2 constant - identical to reach other but diff on diff antibodies
Epitope - binds antigen binding site, binds specific target protein
Produced by all mammals
Best way to use antibodies to label proteins
Use antibodies to attach flouresncet tag to one specific structure
What part of antigen combines with antigen binding sites
Epitopes or antigenic determinants
Most antigens have a variety of theses
What can antibodies be generated against
Most macromolecules - almost all proteins, polypeptides and many polysaccharides
Usually generated against part of peptide sequence of that protein = using synthetic peptide, not full protein
Also can generate against small molecules like aas, and mono amines if conjugated to carrier protein
Can an antibody bind to any size peptide
NO
Cannot be smaller than binding site - cannot recognize
Then needs to be conjugated to carrier
What is immunization
Animals = rabbit, goat, sheep, Guinea pig, Donkey…) = injected at specific intervals with suspension fo antigen conjugated or not to a carrier
Take human protein of interest and inject into rabbit = willl cause immune response
End of immunization
Blood removed from animal, after clotting - serum separated and tested by immunocytochemsirty
Kill animal and isolate antibody
What is limitation of immunization to generate antibodies
Blood of rabbit has mixture of antibodies
Binds diff parts target protein
Polyclonal - can chemically modify it, many diff from one B cell, affinity purification
Describe new antibody characterization
If satisfactory immunostaining obtained = new antibody must be characterized = ensure that it recognizes antigen and not something else
Cannot do this with all proteins
When does immunostaining occur
Should not occur if antibody is pre incubated with antigen prior to being applied to tissue sections
What should antibody recognize - new antibody characterization
Should not recognize other antigens with peptide sequence similar to that of antigen - would not be good = increase Risk of off target binding - less specific so more off target background noise = extraneous binding
= if good antibody produced = animal has to receive periodic injections of antigen
Describe why new antibody characterization might not work
Trail and error - if get too similar, human and rabbit proteins too similar - won’t have good immun response
Could inject again = produce more intense immune repsosne but not very effective
Describe antibodies generated by animals - antibody characterization
Polyclonal antibodies - anti sera
= result of activity of several clones of lymphocytes/plasma cells in animals
Often recognize more than one epitope in antigen molecule
Describe monoclonal antibodies
Recognize only one epitope - produce of hybrid myeloma cell line - hybridoma
B cells - in spleen, once activated produce one antibody for life
Describe hybridoma - what is
1980s - for research and therapies
Tried to do make monoclonals with B cells but issue = wont divide forever so made hybdrioma
What are monoclonal antibodies - form what
Frankenstein cells - myeloma cells with B cells
Product of hybridomas - result of myeloma cell line with lymphocytes form an animal - immunized with antigen
Describe Myeloma cell liens
Available for production of monoclonal antibodies - azaguanine resistant = cannot survive in special tissue culture medium called Hat medium
What animals can monoclonal antibodies be generated form
Need cell liens with characteristics - myeloma and B cell = monoclonal antibodies can only come from rats and mice
Describe hybridoma cells
Maintain capacity to grow easily and indefinitely in tissue culture from myeloma and gain from lymphocytes - capacity to produce desired antibody b
Preparation of monoclonal antibodies - 1
Rats/mice immunized with antigen
Animals bled and sera tested by immunocytochemistry
Animals that produce best polycolonal antibodies selected for fusion and further immunized
At end immunization = lymphocytes from animal fused with myeloma cells
Preparation of monoclonal antibodies - 2
Elimination of non fused cells - following fusion cells are grown in hat medium - that kill og myeloma cells, only fused cells survive
Fused cells grown in wells of tissue culture plates in normal medium - and the antibody production tested from spent culture supernatant of each well
Preparation of monoclonal antibodies - 3
At first each well has more than one clone of hybridoma
Cloning - hybridoma cells plated in wells so that an average of one cell per well obtained - once cells grow, supernatant of wells tested, desired clones selected, expanded and cloned again
Production of monoclonal antibodies = whole process
Inject protein of interest into mouse, take spleen of mouse - then fuse B cells - by shocking them = fuse with myeloma cells (cancer cells, can divide forever)
Want to produce antibody forever
Culture on hat medium - lack enzyme of survival on hat so myeloma cells die
B cels will die out too - crisis, competed out of the mixture =
ONLY LEFT WITH hybridoma cells
Divide media so cells v far apart - and put into well, so each well has one B cell fusion
Tricky process -will know if more than one cell in well bc wil see if antibodies binding diff proteins
Break apart target protein and test to see of antibody binds to specific parts = CANNTO GUARANTEE ITLL EVEN WORK
Advantages of monoclonal
One generated = can be obtained in high amounts at low cost
Hybridomas = immortal cell lines ( for polyclonals = animals that produce polyclonal die and need to start over)
Monoclonals produce highly specific staining - better to avoid background staining, polyclonals often have to be affinity purified to avoid this
Disadvantages monoclonals
Difficult and expensive
What is micro injection
Get antibodies = past cell membrane
Needle with antibodies and inject
Not sued much= use immunoflurescence more
What is immunofluorescence
Chemically modify constant end with fluorescent molecule
What is direct immunofluorescence
Directly stick fluorescent on antibody
What is indirect immunofluorescence
Primary binds protein of interest
Secondary binds primary and is fluorescent
Why would we use indirect immunofluorescence over direct
Can have multiple - secondary with fluorescence for one primary = cna make it easier to see bc more fluorescent molecules
Also secondary less expensive and can use fro all, all secondary per species = the same
Can swap out primary antibody easier = cna change what protein of interest is easily and look at diff things
Describe set up = immunofluorescence
Live cell - antibodies cannot cross membrane
Treat with formaldehyde - kills cell, and cross links everything = bound and freeze in space, so cell retains structure
Permeabiize cell - with detergent = dissolve membranes, treat with mild detergent = produce holes, already fixed cells so wont fall apart
Describe set up = immunofluorescence = direct
After pemeabilize cell = Add primary antibody and binds to target hopefully
Describe set up = immunofluorescence= Indiretc
Add secondary on top of primary
What is disadvantage immunofluorescence
If target cell in it - not on membrane = have to kill cell
Describe methanol - variation of immunofluorescence
Fix/permeabilize with methanol = instead of formaldehyde
Does both fix and permeabilize
Describe skip permeabilization- variation of immunofluorescence
Stain surface of proteins only = if protein of interest on cell
Live cell
Describe gluteraldehyde - variation of immunofluorescence
Use small amount gluteraldehyde for stronger fixation = use less to disrupt cell less
Disadvantage = fluoresncet so produces background noise
Describe tissues - variation of immunofluorescence
Tissues usually sectioned prior to staining - unlike cells in tissue culture
Harder than just one cell - for permeabilize and fixing
Describe multiple fluorescent labels
Can use multiple antibodies.= see multiple things
Look at diff structures in same cell
Describe how to do multiple fluorescent labels
Indirect = each primary must be from diff animal. = bc need signals separate - bind diff organelles
Ned to constant regions not the same - can also do direct immunoflureosnce = avoids this issue
Describe antibodies attached to diff colours
Can be used together
Several proteins cna be tracked at once
Sometimes do not know localization of one protein so compare it to known proteins in same cell
How many colours can be used for immunofluorescence
Up to 3 colours - used together
Sometimes 5+ but harder to do more
Describe immunogold technique
Electron microscopy technique
Chemical attach diff stuff
Any heavy metal works
What is gfp
In naturally floreusncent jelly fish
Gfp fusion proteins = ideal for use in Time lapse imaging and photo bleaching experiments in living cells
Can take gene that encodes gfp and attached to gene of protein of interest and create fusion protein
GREEN fluorescent PROTEIN
How to make gfp
Plasmid grown in bacteria - loop dna
Put into cells = cells start expressing gene and see where gfp is
= purify plasmid, trasnfect mammalian cell, gfp protein fusion expressed after 24hrs - time Delay, not immediate
Examine living cells on heated microscope stage
How to construct gfp tagged proteins
More recent technique bc need to splice dna
Gfp tagging proteins involves creating a new artificial gene in which dna coding sequence for gfp fused to protein
Gene created on a bacterial plasmid and must be introduced into cell
When is gfp easier to use
Much easier to use in tissue culture cells (single cells) than in animals
Harder to stick fusion protein into every cell of whole animal
Advantages gfp
Can be used in living and fixed cells
If dna introduced into cell = gfp tagged protein is produced by cell and already fluorescent
Good for live imaging - movies
Can now be used with other colours of fluorescent proteins - even in combo with immunofluorescence
(C term or n terminal end - which ever)
Disadvantages = gfp
1 = Sometimes = gfp protein doesn’t fold properly or misbehaves Bc have massively increased size of protein
2 = Protein may be present in cell in higher than normal amounts = cannot change how much protein, can messs with transcription a bit, not easy to control, no guarantee that amount of protein cell is making is correct
3 = Endogenous proteins in cell= not visualized (bc has no tag on it, bad bc maybe endogenous protein is already doing function and new incorporated protein is just floating around bc endogenous already doing its function)
4 = Expression of gfp proteins in whole animals = more difficult than in tissue culture cells, bc need to do in embryonic state
When not to use gfp
If do not want to stick foreign dna into cell - not all cells can handle it
