Simple ELISA protocol (For screening monoclonal antibodies)

1
Simple ELISA protocol(For screening monoclonal
antibodies)
1. Coat antigen onto microplate
2. Allow protein adsorption and block unoccupied
sites with neutral protein
3. Add antibody solution (hybridoma supernatant)
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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Antibody-based Protein Array
1. Incubate soluble sample on plate or
membrane-immobilized antibody array
1 2 3 4 5 6
2. Add a mix of labeled soluble antibody
against the same set of antigens
1 2 3 4 5 6
Full set of data
3. Incubate with developing system and
quantitate signal
1 2 3 4 5 6
3
Chromatin Immunoprecipitation Assay (ChIP)
1. Crosslink Protein-DNA complexes in situ
2. Isolate nuclei and fragment DNA (sonication
or digestion)
3. Immunoprecipitate with antibody against
target nuclear protein and reverse crosslinks
4a. Identify protein components of
isolated complexes
4b. Identify DNA sequence by PCR,
cloning and sequencing
MCS
4
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