BIOL3040 Lecture Notes - Lecture 9: Fluorescence Microscope, Ubiquitin, Immunoprecipitation

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7 Feb 2017
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FEBRUARY 6 LECTURE 9
Practice for the Exam
- Goal
o wants us to be challenged during exams
- 4 pages long
- pen preferred
- do’t e afaid to ask a uestio
- keep answers short and use vocabulary
- look at all of the questions before beginning
- could be curved
Experiments
- Seeing a protein and where it is localized
o Fluorescence microscopy
Immunocytochemistry
Fix the cell and then permeabilize membrane with detergent (triton-X) then bind
antibodies to proteins and add a fluorescent antibody that will add to the first
antibody
Live imaging
Tag a protein with GFP
When the cell expresses the protein, you can visualize it through time lapse
imaging
- How to study levels or size of a certain protein
o Western blot
Visualizing on a membrane
Leveldetermine based on the thickness or shade of the bands on a western blot
Locationsize markers are used to determine how big the protein is
- Immunoprecipitation
o Purify a protein
Take a cell (lysate) add antibody that is specific to one protein (use less strong detergent
in the early steps)
Add a bead that is stick and binds only to antibodies
Spin it down, and get rid of extra proteins
Add some buffer and add SDS to denature it and remove the other stuff and run
solution on a western blot
o Looking to see what it binds to and to purify it
- Ubiquitin-Proteosome system
o Major protein degradation pathway
o Damaged protein activates enzymes to add ubiquitin to lysine residues on the protein
Only binds to lysine
o Long string of ubiquitin on a protein signals other proteins to take it to a proteasome which
chops up the damaged protein into amino acids
- Lysosome
o Other form of protein degradation
o Garbage cans to cytosol proteins
- Proteins in the ER
o Glycosylation
Chain of sugars is added to asparagine residues as the polypeptide is threaded through
Once you have the chain of sugars added onto the polypeptide chain, further
modifications occur in the Golgi depending on what enzymes are added
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