Term
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Definition
Many techniques already covered:
SDS-PAGE
Native
Western
2D Gels
This section:
Protein Sequencing
Protease Digestion
Pull downs
Calorimetry
Yeast Two-Hybrid
Surface Plasmon Resonance
Protein-DNA analysis |
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Term
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Definition
N-terminal sequencing via Edman degradation
Samples must be pure (even keratin can contaminate it)
Can select band from gel or blot
50-60 amino acid limit
Mass spectrometry sequencing
Digest protein
Analyze fragments
Repeat with different enzymes
Rebuild protein |
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Term
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Definition
Domain analysis
Fingerprinting
Protein conformation analysis |
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Term
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Definition
| Like CoIP, but bait is a protein instead of an antibody. Uses affinity chromatography (GST, His tag) |
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Term
| Tandem Affinity Purification |
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Definition
To purify proteins with the TAP protocol, apply the mammalian cell lysate to the streptavidin resin, elute using biotin, and apply that eluate to a calmodulin resin.
Once you elute with EGTA, you will get exceptionally pure proteins. |
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Term
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Definition
Identify protein-protein interactions
Essentially a western blot, but with a labeled protein probe instead of an antibody
Native (not SDS) unlike Western
Radiolabeled bait protein (not an antibody) for screening |
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Term
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Definition
Protein-protein interactions
Study known interactions
Find new interactions with a bait
If your two proteins interact, then a reporter gene (lacZ) is transcriptionally activated color change
Bait + binding domain
cDNA library with special plasmids that contain the activating domain built into the system
Interaction allows RNA polymerase to begin transcription color change |
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Term
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Definition
Measurement of heat change when two proteins interact
Types:
Isothermal Titration Calorimetry – measures heat change when adding a peptide to a protein
Differential Scanning Calorimetry – changes temperature and measures results (comparing a protein vs. a mutant)
DG = -RT ln K = DH – TDS
Kd, DH, DG, TDS |
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Term
| Surface Plasmon Resonance |
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Definition
Immobilize protein on membrane
Run protein/peptide across it
Run buffer across it with no protein/peptide
Measure rate of what associates vs. what dissociates to determine dissociation constant (i.e. ‘tightness’ of binding), or in other words saturate the system, then measure the time it takes to dissociate |
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Term
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Definition
Search for proteins that bind DNA
SDS-PAGE, remove SDS, refold with urea, blot onto nitrocellulose
Add digested labeled DNA, look for DNA-binding proteins
Can locate tissue-specific DNA-binding proteins |
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