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Simple ELISA protocol

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.

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Simple ELISA protocol

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  1. 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

  2. ELISA (or Western Blot) • Antigen + antibody bind – wash • Antigen + antibody + anti-antibody-enzyme – wash • Antigen + antibody + anti-antibody- enzyme + substrate • Determine (quantitate) color reaction

  3. Home pregnancy test

  4. ELISA Direct Indirect

  5. Direct and indirect immunofluorescence

  6. Western Blot

  7. Western blot • Look at Western Blot • Protein electrophoresis • Staining protein gel • http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html

  8. Protein electrophoresis

  9. To determine size

  10. Other protein techniques

  11. 2D electrophoresis – 2 steps • Take the isoelectric focusing gel (IEF) • Proteins separated based on charge • Place it on top of the SDS gel • Separated based on size • Run gel – proteins run from the IEF gel into the SDS gel • Stain gel – analyze different spots

  12. 2D-electrophoresis

  13. Picture of a 2D gel

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