Sodium Dodecyl-Sulphate PolyAcrylamide Gel Electrophoresis

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Sodium Dodecyl-Sulphate PolyAcrylamide Gel
Electrophoresis

• Lecture 19

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04_05_Hydrophobic.jpg
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04_29_Disulfide bonds.jpg
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Polyacrylamide
liquid
solid
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Occurrence in daily life

• Acrylamide in fried or baked goods is produced by
  the reaction between asparagine and reducing
  sugars (fructose, glucose, etc.) or reactive
  carbonyls. Browning during baking, frying or
  deep-frying will produce acrylamide and
  over-cooking of foods will produce large amounts
  of acrylamide. Acrylamides can also be created
  during microwaving. Acrylamide cannot be created
  by boiling, and very few uncooked foods contain
  any detectable amounts.
• Acrylamide in olives and prune juice comes
  through another process. It has been suggested
  that environmental pathways, such as the
  breakdown of the herbicide glyphosate (Roundup),
  are sources too.
• Smoking is also a major acrylamide producer.
  Estimates for the proportion in the diet coming
  from the consumption of coffee range from twenty
  to forty percent.

http//en.wikipedia.org/wiki/Acrylamide
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Health Risks

• Potential health risk There is evidence that
  exposure to large doses can cause damage to the
  male reproductive glands. Direct exposure to pure
  acrylamide by inhalation, skin absorption, or eye
  contact irritates the exposed mucous membranes,
  e.g. the nose, and can also cause sweating,
  urinary incontinence, nausea, myalgia, speech
  disorders, numbness, parasthesia, and weakened
  legs and hands. In addition, the acrylamide
  monomer is a potent neurotoxin, causing the
  disassembly or rearrangement of intermediate
  filaments (nerve damage). Ingested acrylamide is
  metabolised to a chemically reactive epoxide,
  glycidamide.
• Cancer According to a 2005 review, acrylamide
  reliably produces various types of cancer in
  experimental mice and rats. However, studies in
  human populations have failed to produce
  consistent results, and it remains unclear
  whether this is due to a reduced risk in a
  natural setting or the methodological
  difficulties inherent in such studies. For
  example, it might be difficult to isolate the
  effects of acrylamide because it is so ubiquitous
  in western diets.

http//en.wikipedia.org/wiki/Acrylamide
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SDS Gel
Stacking Gel
4 acrylamide 12 acrylamide
Resolving Gel
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Protein Gel
Protein Standard
216 kDa 132 kDa 78 kDa 45.7 kDa 32.5 kDa 18.4
kDa
Resolving gel only

• Lanes
• 1Molecular Weight Markers
• Supernatant
• Pellet
• Resin
• Fraction 2
• Fraction 1

Proteins are measured in kiloDaltons 1 amu 1
Dalton 1,000 atomic mass units 1 kiloDalton
(kDa)
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Calculating Protein Size
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Calculating Protein Size
Protein size 20 40 60
80 100 120
Use the graphed protein standard to calculate
unknown protein sizes
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30 mm from top of gel
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In order to run a protein gel, you have to be
able to assemble the apparatus!