Questions and Answers
What are protein arrays equivalent to?
What is a key advantage of using sandwich immunoassays?
High sensitivity
The yeast two-hybrid assay tests if one protein interacts with another using a _______ and _______ domain.
DNA binding, activation
Immunoprecipitation can only capture one type of protein.
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Match the analytical methods to their primary uses:
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What is the main purpose of a pull-down assay?
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What is FRET an acronym for?
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Study Notes
Protein Interaction Studies
- Proteins seldom act alone, usually working in a complex consisting of many proteins.
- Interaction with different proteins leads to different functions.
Analytical Methods
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Protein Arrays (Bio-chips, Protein Chips)
- Equivalent to DNA microarrays
- A series of substrates (proteins, peptides, antibodies, aptamers) are spotted/immobilized on an array
- Sample is added, and binding of proteins in the sample to substrates on the array is detected
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Sandwich Immunoassay
- Capture antibodies with different specificities are immobilized onto a surface
- Binds specific proteins in the sample
- Detected using a second antibody
- Many different proteins can be assayed on the same chip
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Yeast Two-Hybrid Assay
- Used to test if one protein (‘bait’) interacts with other proteins (‘prey’)
- The bait protein is tagged to the DNA-binding domain (DBD) of the yeast GAL4 transcription factor
- A ‘library’ of prey proteins are tagged to the activation domain (AD) of the GAL4 transcription factor
- Interaction between the ‘bait’ and ‘prey’ will bring the DBD and AD of GAL4 together, reconstituting a functional GAL4 transcription factor, leading to the expression of a reporter gene
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Immunoprecipitation
- Used to study protein-protein interactions
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Pull-Down Assays
- Used to study protein-protein interactions
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Fluorescence Resonance Energy Transfer (FRET)
- Used to study protein-protein interactions, nucleic acid-protein interactions, protein-drug interactions, and enzyme-substrate interactions### Immunoprecipitation
- Immunoprecipitation involves capturing a multiprotein complex by using an antibody to one of the proteins in the complex.
- The other members of the complex can then be isolated and identified using Mass Spectrometry (MS) or analyzed using Western Blot.
- Results are then confirmed by doing a second precipitation using an antibody to another protein in the complex.
Pull-Down Assay
- The pull-down assay is similar to immunoprecipitation, but a bait protein is used instead of an antibody.
- The bait protein is tagged with a ligand and immobilized on a solid substrate.
- Other proteins that interact with the bait are captured, and the captured proteins are eluted and analyzed using MS.
Protein Identification
- Proteins can be broken down into smaller peptides using an enzyme like trypsin.
- The peptides can then be analyzed using MS, which can identify the proteins present in the complex.
Fluorescence Resonance Energy Transfer (FRET)
- FRET is a technique used to study protein-protein interactions.
- It involves the non-radiative transfer of energy from an excited fluorophore (donor) to another fluorophore (acceptor).
- This energy transfer only occurs when molecules are in very close proximity (1-10nm).
- FRET can be used to determine when and where two or more molecules, often proteins, interact.
FRET Applications
- FRET can be used to study protein-protein interactions, protein-DNA interactions, and protein conformational changes.
- A common combination of fluorophores used for studying protein-protein interactions is Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP).
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Description
This quiz assesses your understanding of protein interaction studies, their importance, and the learning outcomes related to the topic. It's a complex subject that requires a thorough grasp of protein interactions and their roles in various biological processes.
