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

ELISA (or Western Blot)

• Antigen + antibody bind – wash

• Antigen + antibody + anti-antibody-enzyme – wash

• Antigen + antibody + anti-antibody- enzyme + substrate

• Determine (quantitate) color reaction

Home pregnancy test

ELISA

Direct Indirect

Direct and indirect immunofluorescence

Western Blot

Western blot

• Look at Western Blot

• Protein electrophoresis

• Staining protein gel

• http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html

Protein electrophoresis

To determine size

Other protein techniques

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

2D-electrophoresis

Picture of a 2D gel