Topic 3: Tracers and Probes Flashcards
What are tracers?
Molecules used to view areas in cells(fluorescent dye)
What are Probes?
Molecules that are connected to something like a protein to look in detail to the area of interest.
Radioisotope
atoms that have varying numbers of neutrons that start to get back to the original number and in the process that results in beta particles to come from within.(DNA could be tracked by using the beta particle)
Autoradiography
pictures of black dots on a sample which represents where the radioisotope was because of the beta particles that left the isotope was spotted there.
What is a pulse-chase experiment and how does it work
pictures showing when an isotope is put in a cell and the protein or DNA travel.
Radioactive isotope is put in cell, wait, take pictures at different times to determine where the proteins that are made move
How are antibodies formed
When antigens are present
what type of molecule an antibody is (DNA/protein/lipid/carbohydrate)
protein
the general structure of an antibody
Chain peptides with antigen binding sites on top
how antibodies are made by B cells
B cells are white blood cells that form antibodies when an antigen connects to its B cell.
Explain why antibodies are often used in cellular research.
- inject human protein A into mouse
- Mouse recognizes human protein as foreign
- mouse B cells make a lot of antibodies for human protein A
- take out blood blood
- Then put a flourophore on the mouse antibody to see where the protein moves
Distinguish between primary and secondary antibodies.
Primary antibodies are those that attach to the antigen that is connected to the protein. Secondary antibodies come and connect to the primary antibody and have fluorescent dye which makes it glow a lot and make the protein much more visible.
Describe the characteristics of a fluorescent molecule.
Takes in light and sends out a larger wavelength of light
which antibody has a florurophore when primary and secondary antibodies are present
Secondary Antibody
Explain how a fluorescence microscope is used to produce fluorescent images. Be sure to mention the beam-splitting mirror in your explanation.
A beam splitting mirror is needed for the fluorescent microscope. It lets each wavelength of light to be seen with a colour and the light hits the specimen after hitting the mirror then we see the colours. First filter lets blue light hit mirror (450nm) then hits object with light (510nm) then finally at (560nm) it comes to the eye.
Distinguish between immunogold and immunofluorescence experiments. Make sure to include:
a. What type of tracer is used
b. What type of microscope is used
c. What the final microscope images look like
d. The purpose of each type of experiment
Immunogold tracer:gold particle (black under electron microscope)
immunofluorescence tracer: fluorescence
Immunogold microscope: electron microscope
immunofluorescence microscope: fluorescence microscope
immunogold images: black dots where gold particle is
immunofluorescence pictures: fluorescent where flouorophore
immunogold purpose: can view where proteins go based on density
immunofluorescence purpose: can view where proteins go by amount of light it emits and colour
What is GFP
Green Fluorescent Protein which allows not only pictures but video footage of living thing moving
Describe GFP to include:
a. its source
b. its basic structure
c. the benefits of using GFP over fluorescently-labeled antibodies in the lab
a) came from jellyfish
b) beta sheets and alpha sheets
c) can see live footage
what is a chimera
Protein +GFP
- Explain how an organism that doesn’t normally fluoresce (like a mouse) could be made to fluoresce using GFP.
Insert GFP before or after gene then transcribe and translate the protein and the GFP gets transcribed and translated with it making the organism glow.
How to get better resolution on microscope
Short wavelength = short distance b/w objects therefore better resolution
Transmission Electron Microscope:
2D image that shows inside of cell because electrons go through substance
Scanning Electron microscope
3D image because electron hit the outside of the cell because electrons hit the outside but don’t go through
how do you see darker dots by an electron microscope
If not as many electrons hit the photographic plate then the dots look darker.
