Electrophoresis

  • Created by: LBCW0502
  • Created on: 23-10-18 10:02
What is the name of the physical process underlying separation of DNA molecules in an agarose gel?
Chromatography (separation of sample)
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What is the role of SDS?
Sodium dodecyl sulfate - support medium for proteins (coats proteins with negative charge and breaks down proteins to form linear structures - removes secondary/tertiary/quaternary structures)
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How did Sanger sequencing contributed to the Human Genome Project?
Sanger sequencing used to determine the first human genome but looking at base pairs using primers and templates for sequencing
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What is electrophoresis?
Separation of charged molecules by differences in their rate of migration in an electric field
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Describe features of electrophoresis (1)
Separation based on mobility of ionised particles dissolved in conducting medium under influence of an electric field. Mobility (mu) - average velocity with which ions move in an applied electric field, velocity determined by two opposing forces
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Describe features of electrophoresis (2)
Electrophoresis - Large molecules at the top near the anode, smaller molecules in the middle and smallest molecules at the bottom near the cathode (larger particles/slower movement, smaller particles/faster movement)
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What are the components of the system?
Molecules to be separated (proteins/nucleic acids). Support medium (gel - starch/polyacrylamide/agarose). Buffer system (high buffer capacity, lots of ions, carry charge). DC power source 50-1000V (AC- sample will not move/electrodes change)
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What are the common support media for biological molecules?
Proteins (native gel - acrylamide/starch - suitable for globular proteins/move faster, denaturing SDS gel/acrylamide). Nucleic acids (DNA/RNA) - agarose gel, acrylamide gel
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What are the factors that influence activity?
Properties of the molecules to be separated - MW, shape, charge. Properties of the system - electric field strength (more movement) (V/cm), porosity of the support medium (more holes, more movement) (%S), conductivity of buffer (k), pH of buffer
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How do the factors in electrophoresis interact? (1)
Mobility is proportional to charge/MW of molecule analysed (charge affected by buffer pH). Mobility proportional to field strength (high conductivity/current/heat, field strength increased by V/reduce length of gel)
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How do the factors in electrophoresis interact? (2)
Mobility proportional to buffer conductivity (amount of ions in solution, more ions/current). Strong electrolytes are better conductors (highly dissolved). Proportional to ionic charge >1 charge carry more current
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Electrophoresis systems are designed to optimise what?
Electrophoresis systems designed to optimise separation of specific molecule types based on specific molecular parameters
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Describe features of making a separation - Nucleic Acids
Charge/BP is a constant. Separated can be based on a number of base pairs (given all molecules have the same shape). Larger molecules move slower due to friction with gel
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Describe features of making a separation - Proteins
Charge varies as a function of amino acid composition and buffer pH. Separation is based on charge/MW (shape may also vary). Exact combination of factors varies for each molecule
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Describe the separation of DNA molecules in agarose gels
Molecules are linear. Phosphate groups bear negative charge at neutral pH (2 phosphates/BP). Mobility based on number of base pairs/molecules
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Describe features of typical DNA electrophoresis
Agarose gel, TAE buffer (Tris, acetate, EDTA) - alkaline buffer/pH protects against hydrolysis of DNA. 40mM Tris (strong base), 20 mM (weak acetic acid), 1 mM EDTA (ion chelator, collects Mg ions for DNAase)
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Give an example of an agent used for nucleic acid staining
Ethidium bromide - DNA interchelator (inserts itself between base pairs in a double helix)
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Which two factors must be balanced to optimise electrophoresis?
Current and resolution
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Describe features of separating proteins based on MW in SDS-PAGE gels (1)
Too many separation parameters for proteins using agarose gels. Use SDS gels. Proteins heated/denatured to make shape linear (disulfide bonds reduced using dithiothreitol/beta mercaptoethanol).
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Describe features of separating proteins based on MW in SDS-PAGE gels (2)
Proteins coated with ionic detergent (SDS) for negative charge. Separation based on MW alone
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Describe features of polyacrylamide gel
Various available (4-12%), variable gradient (4-12%). Allows for large molecule at top (low %) and small molecules at bottom (high %)
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Outline SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Add SDS to membrane fragments. Place sample in apparatus (gel in glass plates). Current added (electrodes - cathode/anaode). separation takes place (larger peptides/top, smaller peptides/bottom). Remove gel/stain for protein. Identify samples
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What are the downstream detection methods?
More varied than DNA/RNA. In-gel staining (Coomassie blue staining, silver stain). Transfer PVDF membrane for Western blotting (antibody mediated detection for specific proteins)
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What is capillary zone electrophoresis?
Electrophoresis carried out using narrow-bore capillaries (25-125 micrometres internal diameter). Uses high electric field (large SA:Vol, efficient/rapid dissipation of heat) - short analysis times, high peak efficiency - used for DNA sequencing
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Describe the basics of a CZE set up
Source vial (anode/buffer), sample vial (anode), destination vial (buffer/cathode), computer, high voltage supply, capillary (electroosmotic flow from anode to cathode)
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Describe features of capillary electrophoresis chromatography
Non-chromatographic (different information). Small sample size required. Low cost (purchase/disposal). Ideal for compounds that present costly and time consuming challenges in chromatography (e.g. proteins/nucleic acids/chirals). Easy to develop
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Do neutral compounds separate?
Neutral compounds do not separate effectively
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Do negatively-charged compounds travel towards the negative pole?
Negatively charged compounds travel to the anode (positively charged electrode)
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Describe features of electroosmotic flow (1)
Motion of liquid induced by applied potential across porous material, capillary tube, membrane, microchannel or any other fluid conduit. Little effect when electrical double layer < width of channel. Significant in capillary
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Describe features of electroosmotic flow (2)
Caused by static electrically charged particles in solution when electric field is applied. Chemical equilibrium between solid surface/electrolyte solution, interface, net fixed electrical charge, layer of mobile ions (electric double layer)
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Describe features of electroosmotic flow (3)
When an electric field is applied to fluid (electrode), net charge in electrical double layer is induced to move by resulting Coulomb force - resulting flow (electroomostic flow)
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Describe features of electroosmotic flow (4)
Driving force in CZE from electrical double layer exists at liquid-solid interface between capillary wall and bulk of liquid (diagram of electrical double layer). Plug-like (no parabolic flow - high efficiency compound separation/resolution of peaks)
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Describe features of DNA sequencing (application of CZE)
Sanger sequencing (80s-2000s). Fredrick Sanger won 2nd Nobel prize for work. Originally using dideoxynucleotides to produce terminate polymerase elongation. Sanger method used to produced first human genome project in 2001 (Chromosome 22) - HGP
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Sodium dodecyl sulfate - support medium for proteins (coats proteins with negative charge and breaks down proteins to form linear structures - removes secondary/tertiary/quaternary structures)

Card 3

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How did Sanger sequencing contributed to the Human Genome Project?

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Card 4

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What is electrophoresis?

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Card 5

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Describe features of electrophoresis (1)

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