Tracers & Probes Flashcards
What are tracers and probes used for?
To visualize components of a cell
Describe tracers and give an example
Visible molecules that can be localized and monitored for a length of time
ex. fluorescent dye, short-acting radioisotopes, gold
Describe probes and give an example
More specific tracers that are linked to a SPECIFIC molecule (DNA, RNA, proteins)
ex. fluorescent antibody, gold-linked antibody
What are the 6 tracers and probes?
Radioisotopes autoradiography antibodies fluorescence immunogold EM GFP
what is an isotope?
atoms with the same/normal number of protons but a different number of neutrons are isotopes of one another
When is an isotope radioactive?
if it contains an unstable combination of protons and neutrons
What will happen to a radioactive isotope?
It will eventually disintegrate in order to reach a more stable configuration
What happens as a radioactive isotope decays?
it releases particles or electromagnetic radiation that can be monitored
What is the half life of a radioisotope?
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
What are the 3 main forms of radiation that are released from a radioisotope?
alpha particles
beta particles
gamma radiation (photons)
Which is the most common form of radiation released by a radioisotope?
Beta particles
equivalent to one electron
How are radioisotopes used for studies?
beta emitters can be tagged to a specific molecule (nucleic acids or amino acids) and tracked
What is autoradiography?
A technique used to determine WHERE a particular isotope/something is located within a cell
How does autoradiography work?
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
Give an example of when you would use autoradiography
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
How did autoradiography help understand the endoplasmic reticulum?
Through pulse chase experiments
Pulse with isotope-labelled molecule then chase in isotope-free medium
Why are antibodies used as a technique?
To visualize proteins in a cell by tagging them with something (antibodies) we can see in a microscope
What are two techniques that use antibodies?
Antibodies linked to fluorescent dyes
Antibodies linked to gold particles
What is immunofluorescence?
a technique to visualize proteins in a cell that involves antibodies linked to fluorescent dyes
What is immunogold electron microscopy?
A technique to visualize proteins in a cell that involves antibodies linked to gold particles
What kind of microscope does immunofluorescence use?
fluorescent microscope
How are antibodies produced?
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
What do antibodies bind to?
Specific antigens
Describe antibodies
Proteins that bind very tightly to their targets (specific antigens)
produced in vertebrate immune systems to defend against infection
Describe antigens
A foreign substance in the body that triggers the immune response of producing antibodies that will target and bind with the antigen
Describe the basic structure of antibodies
Y shaped proteins Each antibody has: 2 identical heavy chain peptides 2 identical light chain peptides 2 identical binding sites
What happens when an antigen binds to the receptor on an antibody?
The B cell that has the antibody that bonded with an antigen will divide and secrete large amounts of the same antibody
How would a researcher label a protein to fluoresce?
Make the protein of interest serve as an antigen
What are the two types of antibodies?
polyclonal
monoclonal
Describe the process of studying a protein with polyclonal antibodies
- inject protein of interest into live research creature (ex. rat, rabbit) so it becomes the antigen
- B cells will produce antibodies for that antigen and secrete them into blood
- many different B cells will be stimulated to produce antibodies for the antigen
- 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
Describe the process of studying a protein with monoclonal antibodies
- inject protein of interest into research creature
- remove a spleen cell
- fuse spleen cell with a tumour cell in a petri dish
- 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)
What is the purpose of fusing a spleen cell and tumour cell when using monoclonal antibodies?
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
What is the major difference between polyclonal and monoclonal antibodies?
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
How can you get a stronger fluorescence signal?
if you use two antibodies at once
Explain how using two antibodies works
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
Describe fluorescence
A molecule that absorbs light at a specific wavelength and emits light at a longer wavelength
What is a fluorophore?
A fluorescent dye
What are some common examples of fluorophores?
Fluoroescein = emits green
rhodamine = emits red
DAPI = emits blue
What else does fluorescence usually, but not always, involve?
Antibodies
Describe how a fluorescence microscope works
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
Using an immunogold EM, how would you visualize different proteins in the same sample?
Using different sizes of gold particles
What is GFP?
A protein isolated from jellyfish that fluoresces naturally with blue/green light
What is the purpose of GFP?
Allows for REAL time observation of LIVING specimen movement
T or F: GFP is a fluorophore
FALSE it is a protein
How does GFP work?
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
What is the difference between antibody fluorescence and GFP?
Antibodies do not let you watch the movement of proteins in real time
GFP allows you to watch movement of proteins in real time
T or F: GFP has no effect on the protein it is translated with
True, the protein continues to function as normal
Describe a chimera
When GFP is attached to another protein, collectively it is called a chimera
What will happen to the protein that is attached to GFP?
it will glow green and will be able to be studied under a fluorescence microscope
When would you use GFP?
to follow the movements of proteins
