Tracers & Probes

Question 1

What are tracers and probes used for?

Answer 1

To visualize components of a cell

Question 2

Describe tracers and give an example

Answer 2

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

ex. fluorescent dye, short-acting radioisotopes, gold

Question 3

Describe probes and give an example

Answer 3

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

ex. fluorescent antibody, gold-linked antibody

Question 4

What are the 6 tracers and probes?

Answer 4

Radioisotopes 
autoradiography
antibodies 
fluorescence
immunogold EM
GFP

Question 5

what is an isotope?

Answer 5

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

Question 6

When is an isotope radioactive?

Answer 6

if it contains an unstable combination of protons and neutrons

Question 7

What will happen to a radioactive isotope?

Answer 7

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

Question 8

What happens as a radioactive isotope decays?

Answer 8

it releases particles or electromagnetic radiation that can be monitored

Question 9

What is the half life of a radioisotope?

Answer 9

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

Question 10

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

Answer 10

alpha particles

beta particles

gamma radiation (photons)

Question 11

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

Answer 11

Beta particles

equivalent to one electron

Question 12

How are radioisotopes used for studies?

Answer 12

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

Question 13

What is autoradiography?

Answer 13

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

Question 14

How does autoradiography work?

Answer 14

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

Question 15

Give an example of when you would use autoradiography

Answer 15

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

Question 16

How did autoradiography help understand the endoplasmic reticulum?

Answer 16

Through pulse chase experiments

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

Question 17

Why are antibodies used as a technique?

Answer 17

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

Question 18

What are two techniques that use antibodies?

Answer 18

Antibodies linked to fluorescent dyes

Antibodies linked to gold particles

Question 19

What is immunofluorescence?

Answer 19

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

Question 20

What is immunogold electron microscopy?

Answer 20

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

Question 21

What kind of microscope does immunofluorescence use?

Answer 21

fluorescent microscope

Question 22

How are antibodies produced?

Answer 22

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

Question 23

What do antibodies bind to?

Answer 23

Specific antigens

Question 24

Describe antibodies

Answer 24

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

produced in vertebrate immune systems to defend against infection

Question 25

Describe antigens

Answer 25

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

Question 26

Describe the basic structure of antibodies

Answer 26

``` Y shaped proteins Each antibody has: 2 identical heavy chain peptides 2 identical light chain peptides 2 identical binding sites ```

Question 27

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

Answer 27

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

Question 28

How would a researcher label a protein to fluoresce?

Answer 28

Make the protein of interest serve as an antigen

Question 29

What are the two types of antibodies?

Answer 29

polyclonal monoclonal

Question 30

Describe the process of studying a protein with polyclonal antibodies

Answer 30

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

Question 31

Describe the process of studying a protein with monoclonal antibodies

Answer 31

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)

Question 32

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

Answer 32

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

Question 33

What is the major difference between polyclonal and monoclonal antibodies?

Answer 33

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

Question 34

How can you get a stronger fluorescence signal?

Answer 34

if you use two antibodies at once

Question 35

Explain how using two antibodies works

Answer 35

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

Question 36

Describe fluorescence

Answer 36

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

Question 37

What is a fluorophore?

Answer 37

A fluorescent dye

Question 38

What are some common examples of fluorophores?

Answer 38

Fluoroescein = emits green rhodamine = emits red DAPI = emits blue

Question 39

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

Answer 39

Antibodies

Question 40

Describe how a fluorescence microscope works

Answer 40

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

Question 41

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

Answer 41

Using different sizes of gold particles

Question 42

What is GFP?

Answer 42

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

Question 43

What is the purpose of GFP?

Answer 43

Allows for REAL time observation of LIVING specimen movement

Question 44

T or F: GFP is a fluorophore

Answer 44

FALSE it is a protein

Question 45

How does GFP work?

Answer 45

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

Question 46

What is the difference between antibody fluorescence and GFP?

Answer 46

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

Question 47

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

Answer 47

True, the protein continues to function as normal

Question 48

Describe a chimera

Answer 48

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

Question 49

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

Answer 49

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

Question 50

When would you use GFP?

Answer 50

to follow the movements of proteins