Microscopy II Flashcards

Question

Preparation of monoclonal antibodies - 3

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

Difficult and expensive

Answer 5

Get antibodies = past cell membrane Needle with antibodies and inject Not sued much= use immunoflurescence more

Answer 6

Chemically modify constant end with fluorescent molecule

Answer 7

Directly stick fluorescent on antibody

Answer 8

Primary binds protein of interest Secondary binds primary and is fluorescent

Answer 9

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

Answer 10

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

Answer 11

After pemeabilize cell = Add primary antibody and binds to target hopefully

Answer 12

Add secondary on top of primary

Answer 13

If target cell in it - not on membrane = have to kill cell

Answer 14

Fix/permeabilize with methanol = instead of formaldehyde Does both fix and permeabilize

Answer 15

Stain surface of proteins only = if protein of interest on cell Live cell

Answer 16

Use small amount gluteraldehyde for stronger fixation = use less to disrupt cell less Disadvantage = fluoresncet so produces background noise

Answer 17

Tissues usually sectioned prior to staining - unlike cells in tissue culture Harder than just one cell - for permeabilize and fixing

Answer 18

Can use multiple antibodies.= see multiple things Look at diff structures in same cell

Answer 19

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

Answer 20

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

Answer 21

Up to 3 colours - used together Sometimes 5+ but harder to do more

Answer 22

Electron microscopy technique Chemical attach diff stuff Any heavy metal works

Answer 23

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

Answer 24

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

Answer 25

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

Answer 26

Much easier to use in tissue culture cells (single cells) than in animals Harder to stick fusion protein into every cell of whole animal

Answer 27

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)

Answer 28

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

Answer 29

If do not want to stick foreign dna into cell - not all cells can handle it

Microscopy II Flashcards

(53 cards)