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:
- Check for existing literature on how other scientists have visualized your antigen of interest.
- Check the antibody supplier’s specific antigen retrieval protocol. If no protocol is available, Boster recommends using HIER rather than PIER.
Brief Comparison of HIER and PIER Protocols:
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:
- Microwave/steamer/pressure cooker/waterbath
- 0.01 M citrate buffer solution (pH 6.0)
- 0.01 M PBS buffer (pH7.0)
- 0.05 M EDTA (pH 8.0)
- 0.05 M Tris-EDTA (pH 9.0)
- 0.05 M Tris-HCl (pH 1~12)
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)|