L5 Techniques of Molecular Biology: Analysis of Protein Flashcards
Which is a more stable macromolecule: DNA or protein?
- DNA is more stable
- bc protein conformation/stability is highly susceptible to chemical environment and temperature (usually kept at 4°C)
how do you purify a protein from a cell lysate?
Column chromatography techniques
Column chromatography techniques - What properties of proteins can be used to separate individual molecules?
- ion
- size and shape
- affinity
Column chromatography techniques - Ion-exchange Chromatography
- beads have positive or negative charge to bind complementary proteins
- elute/remove with salt (masks charge and releases interaction)
Column chromatography techniques - Gel-filtration chromatography
- separates protein on the basis of size and shape
- the beads are porous and will capture small protein
- results in small proteins going down slowly and larger ones going down quicker
- collect different elution fractions at different times
Column chromatography techniques - Affinity Chromatography
- attach known substrate or binding molecule to bead
- beads with the attached molecule goes down slower.
What type of gel can be used to physically separate proteins?
Polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis - how does it work?
- heat protein with SDS and β-mercaptoethanol
- Coomassie Brilliant Blue binds and stains the protein nonspecifically
Polyacrylamide gel electrophoresis - heat the protein
- protein is denatured bc SDS removes disulfide bonds
- SDS coats protein and gives it a uniform negative charge
For proteins, what is analogous to nucleic acid hybridization, given that there are no complementary base pairs to exploit?
Exploitation of Immunochemistry (antibodies)
How do you probe for the presence/abundance of a protein?
- Western Blotting
- Immunohistochemistry or Immunofluorescence
Western Blotting - how does it work
- electrophoresis and transfer to a membrane
- antibody detection
how can you identify all proteins present within a cell lystate?
Mass spectrometry (proteomics)
mass spectrometry (proteomics) - how does it identify the protein?
- by its presence (qualitative) and abundance (quantitative)
- produces a graph
mass spectrometry (proteomics) - how do you read the graph?
- individual dots will show more or less expression based on how far they deviate from the line
- more deviation = less expression
how can you test/determine protein-protein interactions?
- “guilt by association” - if you don’t know what it does, figure out what it associates with
- Immunoprecipitation-Mass Spectrometry (IP-MS)
Immunoprecipitation-Mass Spectrometry (IP-MS) - how does it work?
- Have mixture of protein that is isolated in non-chemically harsh way to not disrupt interactions
- Apply antibody and pull out protein of interest (along with its ‘friends’)
- Need to identify friends by using mass spectrometry and will have to separate it via electrophoresis
