Protein Analysis Flashcards
What are biochemical approaches to protein analysis?
Purify protein from a MIXTURE of proteins
and
Identify that protein
Why do we need to purify proteins?
Cells contain ~20,000-30,000 proteins so need to isolate protein of interest
Can run tests on this purified protein to address the structure and function of the protein
Why would we want to identify proteins?
Protein may have a specific activity:
- Antibiotic activity
- Fluorescent protein
- May want to see WHY it has a certain activity - in order to identify similar proteins which may have the same activity
- May want to isolate the activity and use it as a treatment
What is proteomics?
Analysis of the complete protein content in a living system (PROTEOSOME)
INCLUDING ALL post-translationally modified proteins and alternatively spliced variants
Why would you want to carry out proteomics?
To compare the proteins present in one cell to the proteins present in another cell
What does typical protein purification involve?
1) Tissue homogenisation
2) Separation of the released material from unbroken material by centrifugation:
- Ion-exchange
- Gel filtration
- Affinity
3) Several column chromatography steps
4) Confirmation of protein purity after each purification step
What can be used to homogenate tissue?
Sonication
Blending
Pestle and mortar
Describe column chromatography
- The column is packed with a matrix (many little beads)
- The beads have different characteristics (used to separate the proteins)
In column chromatography, what can the proteins be separated by?
Size
Affinity
Ion exchange (separated out due to charge)
What is column chromatography used for?
To separate out proteins, in order to concentrate the protein of interest - can study structure and function
How can protein purity be tested? (4 ways)
- Electrophoresis
- Western immunoblotting
- Mass-spectrometry
- Protein specific assay
What is the process of DIFFERENTIAL centrifugation?
1) Spin cell homogenate at LOW SPEED
- Increase G force on solution
- Organelles settle to the bottom
- Forms a pellet and a supernatant
2) Spin at MEDIUM SPEED
3) Spin at HIGH SPEED
4) Spin at VERY HIGH SPEED
In differential centrifugation, what is contained in pellet 1?
- Whole cells
- Cytoskeleton
- Nuclei
In differential centrifugation, what is contained in pellet 2?
- Mitochondria
- Lysosomes
- Peroxisomes
In differential centrifugation, what is contained in pellet 3?
- Microsomes
- Small vesicles
In differential centrifugation, what is contained in pellet 4?
- Ribosomes
- Viruses
- Large macromolecules
In differential centrifugation, what is contained in the supernatant after spinning the homogenate at very high speed?
Pure cytosol
Highly soluble components
After running DIFFERENTIAL cell centrifugation, what are you left with?
What is the next step
Left with 6 samples - pellets 1-4, final supernatant and the beginning mixture
Next, need to test all the samples for the activity of interest (eg. enzymatic activity if an interested in enzyme)
What is the process of DENSITY-BASED cell centrifugation?
Use a sucrose gradient:
1) Spin the sample, form a gradient of sucrose concentration (high at bottom, low at top)
2) Sample sediments at different levels within the sucrose gradient
3) Empty the sample from the tube at the bottom and collect the liquid
After running DENSITY-BASED centrifugation, what are you left with?
Fractions containing different samples of the liquid and the sediment
In DENSITY-BASED centrifugation, how do the sample sediments settle in the sucrose?
At different concentrations of sucrose, depending upon where the sediments reach equilibrium with the sucrose
(Dense particles at the bottom, where the sucrose is concentrated. Light particles at the top, where the sucrose is at lower concentration)
How is density-based centrifugation better than differential centrifugation?
Can separate out components in ONE round of centrifugation
Describe the process of size-exclusion gel-filtration chromatography
1) Fill the column with porous beads (have holes in with a CONTROLLED SIZE)
2) Pour sample through
3) Small proteins enter bead - are slowed down
Larger proteins do not enter the bead (are EXCLUDED) - have a shorter path and travel faster
4) Collect fractions of the liquid which come out of the column over time (keeping the fractions separate)
Why is buffer continuously added to the top of the column, during liquid chromatography?
To carry the sample through the column