Comparison of electrophoresis between proteins and DNA and RNA

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Comparison of electrophoresis between proteins
and DNA and RNA
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Electrophoresis goes from the negative to the
positive electrode

• DNA and RNA are negatively charged due to the
  phosphate groups
• Proteins are negatively charged because SDS is
  added to the proteins

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SDS serves two purposes

• It makes the protein negatively charged
• It breaks the bonds in the protein so that the
  protein is linear and can move through the gel

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Gel set up DNA

• DNA is usually run in a horizontal position

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DNA sequencing gels are run in a vertical position
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DNA sequencing apparatus
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Protein gels are run in a vertical position
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Running buffer for DNA

• Running buffer must completely cover the gel or
  the gel will not run

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Running buffer for proteins

• Running buffer is needed in the top chamber to
  cover the gel wells and in the bottom chamber to
  cover the bottom of the gel
• The entire tank does not need to have buffer

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Loading buffers are used for DNA and proteins

• Two purposes
• Color allows you to detect the progress of the
  electrophoresis
• The buffer contains a molecule that will weight
  down the sample so that it does not float out of
  the wells

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Example loading buffers for DNA

• I 0.25 bromophenol blue
• 0.25 xylene cyanol FF
• 40 sucrose in water
• II 0.25 (W/V) bromophenol blue
• 0.25 (W/V) xylene cyanol FF
• 30 glycerol in water
• III 0.25 (W/V) bromophenol blue
• 40 (W/V) sucrose in water

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Loading buffers for proteins

• Glycerol
• SDS

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Running buffer for DNA

• TBE
• Tris borate EDTA

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Running buffer for proteins

• Tris
• Glycine
• SDS

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Additional treatment of sample before
electrophoresis

• DNA usually none
• Proteins
• Heat (boil) a sample with a reducing agent
• example reducing agents
• b-mercaptoethanol or dithiothreitol (DTT)
• which further denatures the proteins
• this is known as denaturing electrophoresis
• Sometimes nondenaturing electrophoresis is
  performed

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Composition of gels DNA

• Agarose
• Can have different percentages
• Lower percentage of agarose is needed for larger
  size DNA fragments
• Higher percentage agarose is used for smaller
  size DNA fragments

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Composition of gels protein

• Acrylamide
• Poured between glass plates because oxygen
  inhibits the polymerization process
• Can have different percentages
• Based on size of the proteins

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Different acrylamide for different size proteins
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Staining gels

• DNA
• Ethidium bromide or methylene blue
• Proteins
• Coomassie blue

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How much sample should you use?

• DNA
• About 1 mg of DNA
• You may need more if you have a large gel or if
  you are trying to purify a fragment of DNA
• Protein
• 10-40 mg of protein if you have a mixture of
  proteins
• You can use less protein if you have a purified
  protein

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Viewing gels

• DNA
• If stained with ethidium bromide use UV light
• If stained with methylene blue use white light
• Protein
• View with white light

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White light vs. UV light boxes
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Take pictures to document results
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Picture of DNA gel after staining with methylene
blue
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Picture of DNA gel after staining with ethidium
bromide
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Picture of a protein gel after staining with
coomassie blue
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Determining the size of your DNA or protein

• When you run a gel, always have MW standards in
  one lane
• To determine the MW of your sample, you may be
  able to look at the gel.
• If not, make a graph of the migration of the
  standards and their molecule weights
• Compare the migration of your sample to the
  migration of the standards