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14 Cards in this Set
- Front
- Back
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Why is there a need to purify cellular samples when investigating proteins? |
Many macromolecules are mixed up in the cellular syrup including your protein of interest. The other macromolecules could mess up the results you are trying to collect by influencing the protein. |
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What properties of macromolecule can we exploit when purifying a substance? |
1. Solubility 2. Charge 3. Size 4. Binding affinities |
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How do dialysis bags work for purification? What properties do they purify based on? |
Buffer surrounds a dialysis bag filled with a concentrated mixed solution. Only molecules big enough to remain in the bag / less soluble than the buffer solution are left in the dialysis bag. Exclude based on size or solubility. |
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1. What types of Molecular exclusion gels are there? 2. How does each type of molecular exclusion gel work? 3. What properties do they purify based on? |
1. Size exclusion gel - with uncharged beads , charge exclusion gel with charged beads and binding affinity exclusion gels. 2. Size exclusion - big molecules pass between beads and small beads pass through beads - big molecules first small molecules second. Charge exclusion - same charge molecules pass through and oppositely charge interact and take longer to get through. Binding Affinity - gel is made up of beads bound to small molecules e.g. glucose which interact with the macromolecules which run through the gel - high affinity = slower 3. Charge / Size / Affinity to a particular molecule |
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How does high pressure liquid chromatography work? |
Molecules in a sample mixed with a pressurised liquid solvent passed over a solid adsorbant material. Each compound interacts with the solid material slightly differently , causing different flow rates for each component which can be detected by a reader and computer. |
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What does polyacrylamide gel electrophoresis measure? |
Each different molecule has its mass measured by distance of migration through the gel. |
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What is Isoelectric Focusing (IEF)? |
A separation method based on different isolectric points of different proteins. |
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What is 2-D electrophoresis otherwise known as? |
SDS-PAGE |
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What does SDS-PAGE help to achieve? |
SDS denatures 3-D protein structure and thus will separate proteins purely based on their relative molecular masses. SDS normalises the charge:mass ratio on all proteins. |
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What is the difference between the 'Acivity' and the 'Specific activity' in molecular biology? |
Activity = total molecular activity in a given space Specific Activity = Activity of a specific molecule as a proportion of the total activity in a given space. |
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What does ultracentrifugation achieve? |
Separates molecules according to size and shape through a process called sedimentation. |
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What does the rate of sedimentation depend on? |
1. Mass 2. Density of molecule 3. Density of medium 4. Shape of molecule |
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What does mass spectrometry determine? |
The mass to charge ratio |
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What does the mass to charge ratio help to determine? |
The size and shape of a molecule. |
