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31 Cards in this Set
- Front
- Back
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All protein-protein interactions have 4 key characteristics. State them. |
Qualititively Dissociation constant Stoichiometry On/Off rates |
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The methods we use to investigate protein-protein interactions depends on 5 key issues. State all 5. |
Purity of protein Amount of sample Prior knowledge Value of information Nature of protein |
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There are 11 methods that we can use to investigate protein-protein interactions. State 8 of these. |
Answer can include any 8 of the following: ELISA Immuno/radio absorption Fluorescence methods Surface plasmon resonance Isothermal internal calorimetry Analytical ultracentrifugation Immunoprecipitation Differential scanning fluorometry Co-purification Non-dissociation Mass Spectronomy Biolayer interferometry |
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What does surface plasmon resonance exploit? |
Optical properties of prisms |
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What 3 things are added to the prism to exloit this? |
Noble metal film Monochromatic, polarised light source Flow channel for samples |
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What does the noble metal film create? |
Plasmon wave: which will be out of phase with the reflected light |
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Why will the plasmon wav be out of phase with the reflected light? |
Reflection has interaction with protein and ligand |
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State 3 noble metals. |
Gold, silver and platinum |
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The phase of the plasmon wave depends on what? |
The dialectrics of the media on both sides of the gold |
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State one advantage surface plasmon resonance has over biolayer interferometry. State 2 disadvantages surface plasmon resonance has against biolayer interferometry. |
Higher quality results Takes longer and requires more sample |
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Usually, a SPR run consists of 4 main stages. Stage 1: Load one protein of interest onto the gold Stage 3: Load over a solution containing the second molecule of interest State stages 2 and 4. |
Stage 2: Equilibriate with a buffer Stage 4: Wash with buffer to watch the second molecule elute |
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From the data obtaied from SPR, what can be determined from analysis of the binding curve? What can be determined from the dissociation curve? |
Association rate of the 2 molecule (ka) Dissociation rate of the 2 molecules (kd) |
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A ratio of the ka and kd can give you the? What is this a measure of? |
Dissociation constant (KD) Measure of affinity between the 2 molecules |
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State 4 ways that suppliers can attach proteins to the chip during SPR. |
Covalent cross linking 6* His tag-reversible Streptavidin-biotin complex Place a membrane on the chip |
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State 4 advantages of using SPR to identify protein-protein interactions. |
Answer can include any 4 of the following: Label free Provides ka, kd and KD Requires limited protein, at moderate concentration Can get away with protein that isn't so pure Mainly automated-computer does all the work |
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State 4 disadvantages of using SPR to identify protein-protein interactions. |
Answer can include any 4 of the following: Attaching 1st protein can be difficult Detection limit is 100-200Da, some chemicals are below this Throughput is moderate Can suffer from artefacts Expensive |
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A fluorescent probe is used to detect what? |
The melting point of proteins |
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The probe is usually a hydrophibic dye called? |
SYPRO orange |
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The fluorescence of the tag increases when the tag is bound to what part of the protein? |
Hydrophobic parts of the protein |
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When would the fluorescence decrease? |
When the protein is denatured |
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The method uses a standard qPCR machine, however something is added on top. What is it? |
Optical block to look at fluorecence in each well |
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What defines the curve of the fluorescent curve data? |
I=(A+(B-A)/1+e(Tm-T)/C)) |
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State 3 advantages of using DSF. |
Each experiment requires very little sample Set-up is straightforward Instruments can take 96 samples at once Easy to take wide range of compounds |
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State the 3 main uses for DSF. |
Determine the best buffer for a protein Identify a common binder for a protein Screening libraries of inhibitors |
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State 5 potential problems that are common when using DSF. |
Some proteins show WcbF melting curve Some proteins unfold at very low temperatures Some proteins show multiple unfolding steps Some proteins show binary results with compounds |
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State 4 advantages of using DSF for analysing protein-protein interactions |
Answer can include any 4 of the following: Cheap Quick Easy to set up a process for many samples Speed and ease make silly experiments likely Instrument is cheap and can be used for other methods |
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State 3 disadvantages of using DSF for analysing protein-protein interactions |
Answer can include any 3 of the following: Data processing can be tough with semi-automated Not quantitative Dye may be altering the results Some proteins are essentially impossible to detect |
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Compare SPR and DSF. What advantages does SPR have over DSF? |
Gives more high quality information Works well with protein-protein interactions |
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Compare SPR and DSF. What advantages does DSF have over SPR? |
Fast Requires less compound Doesn't use a lot of sample Deals well with individual binding Deals well with multiple binding |
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What is the main function of Surface Plasmon Resonance? |
Determine On/Off rates |
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What is the main function of Differential Scanning fluorometry? |
Allows rapid screening of of proteins for stabalising agents |
