Lecture 6 Flashcards
What is pulse-chase autoradiography?
Pulse-chase autoradiography refers to the fact that newly synthesized proteins were labelled with radiolabelled tags by radioactive amino acids. These proteins are tracked by incubating it and killing cells at various times to see where the proteins were located.
How can yeast cells be used to identify how proteins are transported in the cell prior to secretion?
Yeast sec mutants selected blocked at various stages of secretion. This approach told us about the particular steps and genes involved in vesicle budding and fusion
How can microscopes be used to visualize protein transport in cells?
Cargo can be imaged while it is moving around via microscopy.
Why is location important in understanding what happens in the cell?
To determine the relationship between structure and function of the protein
What is the resolution of the light microscope?
200nm
What is the resolution of the EM?
less than 1nm at the atomic level.
How are proteins followed within the cell?
Probes are used such as antibodies.
What are some features of antibodies that are understood and help with the formation of probes form them?
Antibodies:
Bind tightly to target.
Targets are called antigens.
Produced in animals as a defense against infection.
4 polypeptide chains; 2 light chains and 2 heavy chains. 1 heavy chain and 1 light chain are variable.
What kind of molecules are used during microscopy to visualize structures?
Fluorescent molecules. These molecules absorb light at one wavelength and emit a different (longer) wavelength.
How are microscopes designed for fluorescence microscopy?
A first barrier filter is used that only permits light with a certain wavelength.
A beam-splitting mirror permits that wavelength to be reflected onto the objective lens and the object, the rest of the beam is transmitted.
Second barrier filter filters out the lower wavelengths to allow the fluorescent proteins to show up.
What is the benefit of using confocal microscopy?
Confocal microscopy uses a laser beam to illuminate
molecules only in the focal plane this results in a sharper image.
Why does the conventional fluorescence microscope show a low resolution image?
The light microscope transmits light from above and below the focal plane of the cell which interfere with what is seen.
What is immuno electron microscopy?
Immunoglobins tagged with electron dense molecules to create easy structures to visualize in the lab. This increases the resolution that can be visualized from electron microscopy.
What is the problem with using dead cells for microscopy?
Cells are dead they don’t show what is happening during life.
Cells are fixed and so some antigens are destroyed.
Protein isn’t moving so the movement of the protein can not be visualized.
What is confocal microscopy?
The use of a laser to focus light on a single point and then repeating this with multiple proteins
