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27 Cards in this Set
- Front
- Back
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State the formulae used to calculate the velocity (V) of an electron. Define what each greek letter is meant to represent? |
V=upE up: electrophoretic mobility E: electric field (volts m^-1) |
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State the equation used to calculate electrophoretic mobility. Define what each numeral is meant to represent. |
up=z/6.Pi.Eta.r z: net charge (signed quantity) Eta: the viscosity of the medium r: hydrodynamic radius |
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The positive terminal is also known as the? What about the negative terminal? |
Positive terminal: Anode Negative terminal: Cathode |
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Electrophoresis allows differential mobility based on what 3 things? |
Mass, charge and hydrodynamic radius |
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2 types of electrophoresis gel are agarose and acrylamide. What is each used for? |
Agarose: DNA 200-5,000 base pairs Acrylamide: DNA up to 500 base pairs and proteins |
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What is N,N1-Methylene-Bis-Acrylamide? |
Cross-linking agent used during the formation of polyacrylamide |
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Within acrylamide a matrix is formed. Proteins ineteract by by mass due to what 2 properties? |
Hole size and hydrophobicity |
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There are 5 applications of native gel electrophoresis. State 4 of these. |
Answer can include any 4 of the following: Determine oligomeric states Protein complexation Gel shift assay Separate proteins based on charge 2D electrophoresis |
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Protein migration is sensitive to 6 different effects. State 4 of these. |
Answer can include any 4 of the following: Changes in charge due to chemical degradation Modified conformations Oligomers Aggregates Binding events Excipients |
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What does PAGE stand for? |
Polyacrylamide gel electrophoresis |
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In native PAGE, when charge and hydrodynamic radius is equal, but mass is a variant, do the largest or the smallest molecules travel the furthers through the gel? |
Smallest |
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In an SDS-PAGE, what does SDS stand for? |
Sodium Dodecyl Sulphate |
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Adding SDS to PAGE now allows proteins to be seperated according to? |
Molecular weight of individual peptides |
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A process called 'Stacking' is used in disk gel electrophoresis. What does this process allow? |
Proteins move slowly through stacked region, allows larger proteins to be seperated more effectively |
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In order to calculate relative mobility of proteins, a tracking dye is added. What 2 properties does the tracking dye have that allow it to act as a standard? |
Fixed charge and constant mobility |
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How is relative mobility calculated? |
Distance travelled by a protein/distance travelled by the dye |
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In order to determine the kDa of an unknown protein, a calibration curve is produced. What lies on the x-axis and the y-axis of this graph? |
x-axis: Relative mobility y-axis: log molecular mass (kDa) |
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During isoelectric focussing, the gel is pre-focussed. An electric current is applied, what happens next? |
Ampholytes will migrate to form a pH gradient across the gel |
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In a 2D gel electrophoresis, what 2 methods of spacial differentiation could be used to seperate proteins on 2 axis (same gel)? |
pH range (isoelectric focussing) Molecular weights (SDS-PAGE) |
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To identify a protein, 2 staining methods can be used. State both. To stain nucleic acids, what chemical is used? |
-Coomassive blue in methanol and acetic acid -Silver Nucleic acids: Ethidium Bromide |
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In an autoradiogram, what is DNA labelled with? |
Radioactive Phosphate 32 |
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In blotting, what are the biomolecules transferred onto? |
Nitrocellulose or nylon filter |
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What is analysed in Southern blotting? How is this acheived? |
DNA fragments analysed by interaction with radioactive labelled DNA probes |
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What is analysed in Northern blotting? How is this acheived? |
RNA dtected using specific radioactive DNA probes |
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What is analysed in Western blotting? How is this acheived? |
Protein detection with specific antibodies |
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In blotting, what is sensitivity controlled by? What is specificity controlled by? |
Sensitivity: smallest possible staining you can measure Specificity: Interaction between antibody and enzyme |
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What is pulsed electrophoresis used for? How does it allow separation? |
Separating large DNA fragments Separates by inertia |
