Detection of harmful epitopes in foods - Qualitative tests
6 important questions on Detection of harmful epitopes in foods - Qualitative tests
Why is blocking important in Immunoblotting?
Which enzyme is used to prepare enzyme-antibody conjugates in Immunoblotting?
Mention 2 types of immunoblotting
- Spot blotting
- Diffusion or Western blotting
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What are 2 advantages of using Western blotting
- The technique allows positive identification of allergenic protein, if the band, stained in the immunoblot, corresponds to a known allergen
- In contrast to ELISA techniques, immunoblotting allows detection of all protein present, if they contain a linear epitope and if they are larger than 3000 Da.
Why are conformational epitopes difficult to detect in Western blotting?
Explain the procedure of Western blotting:
- Load an separate protein samples on SDS-PAGE
- Electrophoretically transfer fractionated proteins onto PVDF membrane
- Block the membrane with neutral protein (BSA or milk casein)
- Incubate the membrane with primary antibody specific to target protein
- Incubate the membrane with HRP-labeled secondary antibody specific to primary antibody
- Incubate the blot with chemiluminescent HRP substrate and expose to film
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