PhD Methods

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Rahna
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244892
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PhD Methods
Updated:
2013-11-05 12:02:05
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Methods
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Methods used and reasons for the reagents used
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  1. western blot
    What are the components of the Laemmli buffer (loading dye)?
    • SDS (sodium dodecyl sulphate)
    • b-mercaptoethanol (BME)
    • bromophenol blue
    • glycerol
    • Tris-glycine at pH 6.8.
  2. Western Blot
    In laemmli buffer...what is the purpose of...
    SDS?
    SDS adds a negative charge to the proteins

    • The amount of bound SDS is relative to the size of the protein, and the proteins have a similar charge to mass ratio.
    • it is an anionic detergent and denatures secondary and non-disulfide-linked tertiary structures.
  3. Western Blot
    In laemmli buffer...what is the purpose of...
    BME?
    -BME is added to prevent oxidation of cysteines

    -to break up disulfide bonds
  4. Western Blot
    In laemmli buffer...what is the purpose of...
    Bromophenol Blue?
    Bromophenyl blue is a dye that is useful for visualizing your sample in the well and tracking its progress through the gel
  5. Western Blot
    In laemmli buffer...what is the purpose of...
    Glycerol?
    Glycerol is much more dense than water and is added to make the sample fall to the bottom of the sample well rather than just flow out and mix with all the buffer in the upper reservoir
  6. Western Blot
    what is the components of the running buffer?
    • Tris
    • Glycine
    • SDS
  7. western blot
    What are the components of an SDS PAGE gel?
    • 30% Acrylamide
    • 10% APS
    • TEMED
    • 0.5M Tris-HCl pH 6.8 for stacking
    • 1.5M Tris-HCl pH 8.8 for resolving
  8. What is coomassie stain?
    • 0.2% coomassie Blue
    • 7.5% glacial acetic acid
    • 50% ethanol
  9. How does the pH contribute to resolving the gel?
    • Laemmli gels are composed of two different gels
    • (stacker and running gel), each cast at a different pH. In addition, the gel
    • buffer is at a third, different pH.  The running gel is buffered with
    • Tris by adjusting it to pH 8.8 with HCl. The stacking gel is also buffered with
    • Tris but adjusted to pH 6.8 with HCl. The sample buffer is also buffered to pH
    • 6.8 with Tris HCl (note all the chloride ions – they will become important in a
    • minute).  The electrode buffer is also Tris, but here the pH is
    • adjusted to a few tenths of a unit below the running gel (in this case 8.3)
    • using only glycine – nothing else. We run our gels at constant voltage.

     

    • So here’s what happens when you turn on the
    • power.  Glycine is a weak acid and it can exist in either of two
    • states, an uncharged zwitterion, or a charged glycinate anion (that is to say,
    • negatively charged). At low pH it is protonated and thus uncharged. At higher
    • pH it is negatively charged. When the power goes on the glycine ions in the
    • running buffer want to move away from the cathode (the negative electrode) so
    • they head toward the sample and the stacking gel. The pH there is low and so
    • they lose a lot of their charge and slow down. Meanwhile, in the stacker and
    • sample the highly mobile chloride ions (which are also negatively charged) move
    • away from the cathode too. This creates a narrow zone of very low conductance
    • (in other words very high electrical resistance) in the top of the stacking
    • gel. Because V=IR almost all of the voltage that you put across the gel (110
    • Volts is typical for stacking) is concentrated in this small zone. The very
    • high field strength makes the negatively charged proteins move forward. The
    • trick, however, is that they can never outrun the chloride ions. If they did
    • they would find themselves in a region of high conductance and very low field
    • strength and would immediately slow down. The result is that all the proteins
    • move through the stacker in a tight band just behind the moving front of
    • chloride ions. Behind them, the pokey glycine ions straggle along as best they
    • can (they do move, but with lower mobility than the chloride ions).

     

    • The effect of this moving zone of high voltage is
    • that all the proteins reach the running gel at virtually the same time so that
    • migration of the proteins is truly a function of molecular size and not some
    • complicated function of how carefully you loaded the gel and when you started
    • the voltage.Laemmli gels are composed of two different gels
    • (stacker and running gel), each cast at a different pH. In addition, the gel
    • buffer is at a third, different pH.  The running gel is buffered with
    • Tris by adjusting it to pH 8.8 with HCl. The stacking gel is also buffered with
    • Tris but adjusted to pH 6.8 with HCl. The sample buffer is also buffered to pH
    • 6.8 with Tris HCl (note all the chloride ions – they will become important in a
    • minute).  The electrode buffer is also Tris, but here the pH is
    • adjusted to a few tenths of a unit below the running gel (in this case 8.3)
    • using only glycine – nothing else. We run our gels at constant voltage.

     

    • So here’s what happens when you turn on the
    • power.  Glycine is a weak acid and it can exist in either of two
    • states, an uncharged zwitterion, or a charged glycinate anion (that is to say,
    • negatively charged). At low pH it is protonated and thus uncharged. At higher
    • pH it is negatively charged. When the power goes on the glycine ions in the
    • running buffer want to move away from the cathode (the negative electrode) so
    • they head toward the sample and the stacking gel. The pH there is low and so
    • they lose a lot of their charge and slow down. Meanwhile, in the stacker and
    • sample the highly mobile chloride ions (which are also negatively charged) move
    • away from the cathode too. This creates a narrow zone of very low conductance
    • (in other words very high electrical resistance) in the top of the stacking
    • gel. Because V=IR almost all of the voltage that you put across the gel (110
    • Volts is typical for stacking) is concentrated in this small zone. The very
    • high field strength makes the negatively charged proteins move forward. The
    • trick, however, is that they can never outrun the chloride ions. If they did
    • they would find themselves in a region of high conductance and very low field
    • strength and would immediately slow down. The result is that all the proteins
    • move through the stacker in a tight band just behind the moving front of
    • chloride ions. Behind them, the pokey glycine ions straggle along as best they
    • can (they do move, but with lower mobility than the chloride ions).

     

    • The effect of this moving zone of high voltage is
    • that all the proteins reach the running gel at virtually the same time so that
    • migration of the proteins is truly a function of molecular size and not some
    • complicated function of how carefully you loaded the gel and when you started
    • the voltage.
  10. To resolve proteins, SDS is used. what chemical denaturant is used to resolve nucleic acids?
    • Urea
    • breaks the hydrogen bonds between the base pairs of the nucleic acid, causing the constituent strands to separate.
  11. why heat the samples to 100 degrees Celcius before loading onto gel?
    Denatures the proteins so that the protein folding does not effect the movement of the protein through the gel
  12. what else can be used to denature the proteins?
    • DTT- dithiothreitol
    • BME
    • reduces the disulfide linkages thus overcoming some forms of tertiary protein folding and breaking up quaternary protein structure (oligomeric subunits). This is known as reducing SDS-PAGE.
  13. what is the purpose of adding APS and TEMED to the SDS gel?
    initiate polymerisation
  14. How does the gel cross-link?
    The polymerization reaction creates a gel because of the added bisacrylamide, which can form cross-links between two polyacrylamide molecules.
  15. what is the purpose of acrylamide?
    • When dissolved in water, slow, spontaneous autopolymerization of acrylamide
    • takes place,joining molecules together by head on tail fashion to form long
    • single-chain polymers. The presence of a free radical-generating system
    • greatly accelerates polymerization. This kind of reaction is known as Vinyl addition polymerisation. A solution of these polymer chains becomes viscous but does not form a gel, because the chains simply slide over
    • one another. Gel formation requires linking various chains together. Acrylamide
    • is a neurotoxin. It is also essential to store acrylamide in a cool dark and dry place
    • to reduce autopolymerisation and hydrolysis.
  16. what is the purpose of bisacrylamide?
    Bisacrylamide is the most frequently used cross linking agent for polyacrylamide gels. Chemically it can be thought of as two acrylamide molecules coupled head to head at their non-reactive ends. Bisacrylamide can crosslink two polyacrylamide chains to one another,thereby resulting in a gel.
  17. what does APS do?
    APS is a source of free radicals and is often used as an initiator for gel formation. An alternative source of free radicals is riboflavin, which generated free radicals in a photochemical reaction.
  18. what does TEMED do?
    TEMED stabilizes free radicals and improves polymerization. The rate of polymerisation and the properties of the resulting gel depend on the concentrations of free radicals. Increasing the amount of free radicals results in a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in gel elasticity. Decreasing the amount shows the reverse effect. The lowest catalytic concentrations that allow polymerisation in a reasonable period of time should be used. APS and TEMED are typically used at approximately equimolar concentrations in the range of 1 to 10 mM.
  19. what is PCR?
    • polymerase chain reaction
    • a process used to amplify a single or few copies of DNA across several orders of magnitude.
    • Generating thousands to millions of copies of a particular DNA sequence

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