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BIOL 2321 - Cell Biology > Tracers & Probes > Flashcards

Tracers & Probes Flashcards

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1
Q

What are tracers and probes used for?

A

To visualize components of a cell

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2
Q

Describe tracers and give an example

A

Visible molecules that can be localized and monitored for a length of time

ex. fluorescent dye, short-acting radioisotopes, gold

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3
Q

Describe probes and give an example

A

More specific tracers that are linked to a SPECIFIC molecule (DNA, RNA, proteins)

ex. fluorescent antibody, gold-linked antibody

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4
Q

What are the 6 tracers and probes?

A 
Radioisotopes 
autoradiography
antibodies 
fluorescence
immunogold EM
GFP
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5
Q

what is an isotope?

A

atoms with the same/normal number of protons but a different number of neutrons are isotopes of one another

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6
Q

When is an isotope radioactive?

A

if it contains an unstable combination of protons and neutrons

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7
Q

What will happen to a radioactive isotope?

A

It will eventually disintegrate in order to reach a more stable configuration

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8
Q

What happens as a radioactive isotope decays?

A

it releases particles or electromagnetic radiation that can be monitored

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9
Q

What is the half life of a radioisotope?

A

a measure of its instability

the more unstable an isotope is, the more likely it will disintegrate in a given time period

the more stable it is, the longer it will take to disintegrate

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10
Q

What are the 3 main forms of radiation that are released from a radioisotope?

A

alpha particles

beta particles

gamma radiation (photons)

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11
Q

Which is the most common form of radiation released by a radioisotope?

A

Beta particles

equivalent to one electron

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12
Q

How are radioisotopes used for studies?

A

beta emitters can be tagged to a specific molecule (nucleic acids or amino acids) and tracked

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13
Q

What is autoradiography?

A

A technique used to determine WHERE a particular isotope/something is located within a cell

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14
Q

How does autoradiography work?

A

The particle emitted from a radioactive atom activates a photographic emulsion containing silver halide crystals in gelatin

The photographic emulsion is brought into close contact with the radioactive source and the particles emitted by the source leave TINY SILVER GRAINS (shown in black) in the emulsion after developing the photo

the black dots are where the radiation occurred

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15
Q

Give an example of when you would use autoradiography

A

When looking for the location of uridine in RNA where transcription occurs in the chromosome

the black spots will show where transcription using uridine happened

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16
Q

How did autoradiography help understand the endoplasmic reticulum?

A

Through pulse chase experiments

Pulse with isotope-labelled molecule then chase in isotope-free medium

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17
Q

Why are antibodies used as a technique?

A

To visualize proteins in a cell by tagging them with something (antibodies) we can see in a microscope

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18
Q

What are two techniques that use antibodies?

A

Antibodies linked to fluorescent dyes

Antibodies linked to gold particles

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19
Q

What is immunofluorescence?

A

a technique to visualize proteins in a cell that involves antibodies linked to fluorescent dyes

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20
Q

What is immunogold electron microscopy?

A

A technique to visualize proteins in a cell that involves antibodies linked to gold particles

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21
Q

What kind of microscope does immunofluorescence use?

A

fluorescent microscope

22
Q

How are antibodies produced?

A

Produced by the immune system

made by B lymphocytes (B cells), a type of white blood cell

B cells have many different antibodies on their surface

23
Q

What do antibodies bind to?

A

Specific antigens

24
Q

Describe antibodies

A

Proteins that bind very tightly to their targets (specific antigens)

produced in vertebrate immune systems to defend against infection

25
Q

Describe antigens

A

A foreign substance in the body that triggers the immune response of producing antibodies that will target and bind with the antigen

26
Q

Describe the basic structure of antibodies

A 
Y shaped proteins 
Each antibody has:
2 identical heavy chain peptides
2 identical light chain peptides
2 identical binding sites
27
Q

What happens when an antigen binds to the receptor on an antibody?

A

The B cell that has the antibody that bonded with an antigen will divide and secrete large amounts of the same antibody

28
Q

How would a researcher label a protein to fluoresce?

A

Make the protein of interest serve as an antigen

29
Q

What are the two types of antibodies?

A

polyclonal

monoclonal

30
Q

Describe the process of studying a protein with polyclonal antibodies

A
  1. inject protein of interest into live research creature (ex. rat, rabbit) so it becomes the antigen
  2. B cells will produce antibodies for that antigen and secrete them into blood
  3. many different B cells will be stimulated to produce antibodies for the antigen
  4. an assortment of antibodies will be isolated from the serum and each will bind the antigen in a slightly different way because each antigen will be slightly different
31
Q

Describe the process of studying a protein with monoclonal antibodies

A
  1. inject protein of interest into research creature
  2. remove a spleen cell
  3. fuse spleen cell with a tumour cell in a petri dish
  4. tumour cell will divide indefinitely without producing antibody and spleen cell will make antibodies

resulting in a Petri dish with lots of identical antibodies (all bind to antigen in same way at same location)

32
Q

What is the purpose of fusing a spleen cell and tumour cell when using monoclonal antibodies?

A

the tumour cell will divide indefinitely in the Petri dish but will not produce antibodies

the spleen cell will produce antibodies but not divide indefinitely

Using both allows a Petri dish of many IDENTICAL antibodies

33
Q

What is the major difference between polyclonal and monoclonal antibodies?

A

Polyclonal antibodies will target the same antigen but have different binding sites or different locations for the binding site

Monoclonal antibodies will all target the same antigen and be completely identical in where they bind and how

34
Q

How can you get a stronger fluorescence signal?

A

if you use two antibodies at once

35
Q

Explain how using two antibodies works

A

when antigen detected

primary antibody (ex. rat antibody) will target and bind with the antigen

Secondary antibodies are marked and will target and bind with the primary antibodies

Result = more significant signal tracker, cheaper, and easier

36
Q

Describe fluorescence

A

A molecule that absorbs light at a specific wavelength and emits light at a longer wavelength

37
Q

What is a fluorophore?

A

A fluorescent dye

38
Q

What are some common examples of fluorophores?

A

Fluoroescein = emits green

rhodamine = emits red

DAPI = emits blue

39
Q

What else does fluorescence usually, but not always, involve?

A

Antibodies

40
Q

Describe how a fluorescence microscope works

A

It has filters at key positions to block unwanted wavelengths of light

Also has a beam splitting mirror that will reflect and transmit light at different wavelengths

The object will be hit with one wavelength and fluoresce at the new, longer wavelength

41
Q

Using an immunogold EM, how would you visualize different proteins in the same sample?

A

Using different sizes of gold particles

42
Q

What is GFP?

A

A protein isolated from jellyfish that fluoresces naturally with blue/green light

43
Q

What is the purpose of GFP?

A

Allows for REAL time observation of LIVING specimen movement

44
Q

T or F: GFP is a fluorophore

A

FALSE it is a protein

45
Q

How does GFP work?

A

it manipulates the DNA and places in coding for the GFP protein next to the coding region of a protein of interest so that it will translated with the protein and the protein will become green

46
Q

What is the difference between antibody fluorescence and GFP?

A

Antibodies do not let you watch the movement of proteins in real time

GFP allows you to watch movement of proteins in real time

47
Q

T or F: GFP has no effect on the protein it is translated with

A

True, the protein continues to function as normal

48
Q

Describe a chimera

A

When GFP is attached to another protein, collectively it is called a chimera

49
Q

What will happen to the protein that is attached to GFP?

A

it will glow green and will be able to be studied under a fluorescence microscope

50
Q

When would you use GFP?

A

to follow the movements of proteins

BIOL 2321 - Cell Biology (26 decks)

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