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Here are some technical tips for optimizing your antigen retrieval method!
Formaldehyde fixation usually generates methylene bridges which cross-link proteins and therefore mask the epitope of interest. It is essential to unmask the antibody epitopes in order to allow the antibodies to bind, either by heat (Heat Induced Epitope Retrieval: HIER) or enzymatic digestion (Proteolytic Induced Epitope Retrieval: PIER). To find the optimal antigen recovery method, we suggest that you test both HIER and PIER methods, compare their results and optimize the method as needed. Here are some tips to do this:
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The HIER method can be implemented by microwave, high pressure or water bath. It breaks the methylene bridges and exposes the epitope to allow the antibodies to bind by continuously heating. Compared to PIER, HIER has a gentler experimental condition in which users have more control over the experimental parameters. However, the pH and buffers for HIER must be optimized and determined for each new antibody/antigen target. The following antigen retrieval reagents are recommended:
Epitopes can be exposed by incubation with proteases which can break the methylene bridges. The choice for digestion enzymes depends on the antigenic components. PIER is suitable for retrieving more difficult epitopes while the pH for incubation is usually known. However, PIER is a harsher method and can damage tissue morphology.
Digestion Enzymes for PIER:
|Enzyme||Working Concentration||Digestion Condition|
|Trypsin||0.05% to 0.1%||37°C (10 to 40 min)*|
|Proteinase K||20 µg/mL||37°C (20 min)|
|Pepsin||0.4%||37°C (30 to 180 min)|
* The reaction time can be increased for certain worn-out tissues. Fresh trypsin solution should be prepared with pH adjusted to 7.6 and used at 37°C
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|Applications||IHC, PIER/EIER, PEFF tissues|
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Review: "This is an excellent antibody to endoplasmin in HC11 cells. Clean, reliable detection with very little if any background. Great antibody for Westerns. Could probably be optimized for 1 hour primary incubation."
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