Simple ELISA protocol

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Simple ELISA protocol
1. Coat antigen onto microplate
2. Allow protein adsorption and block unoccupied
sites with neutral protein
3. Add antibody solution into each well
4. Add HRP or AP conjugated secondary antibody
into each well and develop colorimetric
reaction with appropriate substrate
5. Read absorbance in spectrophotometer with
appropriate filter and quantitate relative
antigen levels
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ELISA (or Western Blot)

• Antigen antibody bind wash
• Antigen antibody anti-antibody-enzyme wash
• Antigen antibody anti-antibody- enzyme
  substrate
• Determine (quantitate) color reaction

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Direct and indirect immunofluorescence
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Western Blot
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Western blot

• Look at Western Blot
• Protein electrophoresis
• Staining protein gel
• http//www.bio.davidson.edu/courses/genomics/metho
  d/Westernblot.html

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Protein electrophoresis
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To determine size
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Other protein techniques
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2D electrophoresis 2 steps

• Take the isoelectric focusing gel (IEF)
• Place it on top of the SDS gel
• Run gel proteins run from the IEF gel into the
  SDS gel
• Stain gel analyze different spots

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2D-electrophoresis
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Picture of a 2D gel
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Antibody-based Protein Array
1. Incubate soluble sample on plate or
membrane-immobilized antibody array
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2. Add a mix of labeled soluble antibody
against the same set of antigens
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Full set of data
3. Incubate with developing system and
quantitate signal
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