Epitope Recovery Buffers
Epitopes of varying nature may necessitate the use of different recovery buffers to achieve optimal results. In the case of formalin-fixed/paraffin-embedded (FF/PE) sections, the majority of commercial antibodies, which are typically designed against peptide epitopes or bacteria-expressed protein fragments, can be effectively unmasked using our R-Universal buffer.
However, we have discovered specific instances where, in addition to R-Universal, the use of alternative buffers yields even better outcomes. The choice of the appropriate buffer for epitope recovery depends on the epitope's structural characteristics and the specific amino acids that compose it. Therefore, utilizing different buffers tailored to the epitope's structure can enhance the quality of the recovery process.
The type of epitope defines the most appropriate recovery buffer
As previously mentioned, many commercial antibodies available today are generated against bacterially expressed protein fragments or synthetic peptide epitopes. The standard approach in antibody development involves selecting well-exposed surface epitopes or unique protein fragments for antibody generation. However, in cases where the antibody is targeting a unique mutated or modified epitope, or an epitope that differs among closely related proteins within the same family, the available choices for the epitope may be limited. Such epitopes may not be exposed or may only be present in a mature, folded protein structure.
In these particular cases, R-Universal or standard buffers like EDTA may not be the most suitable options. The chart provided below can assist in selecting the most appropriate buffer for epitope recovery based on the type of epitope:
This epitope is usually recognised by antibodies raised against a hydrophilic peptide or bacterially produced fragments of a target protein.
Discontinuous epitope requires at least some proper folding of the molecule. Antibodies to such epitopes are usually raised against native antigen, often glycosylated, or in complex with other proteins.
Structural epitope requires proper folding of the native protein, usually with cysteine bridges being intact.
In mature protein such epitope is usually hidden. Usually antibody is raised against an immunogenic
but not highly hydrophilic peptide epitope..
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We provide a total of five different buffers for antigen recovery. The majority of epitopes can be effectively recovered using our R-Universal buffer, which includes those typically recovered using Citrate/EDTA/Tris-EDTA buffers. Additionally, we have introduced two new buffers that address rare cases where an antibody produces an incorrect staining pattern on a retrieved section or fails to provide any staining at all.
Furthermore, we now offer analogues of the standard Low (Citrate) and High (EDTA) pH-buffers. These analogues are particularly useful if you already use antibodies that are known to work well when tissues are processed in one of these specific buffers.
Below, we present an additional chart to assist you in selecting the most suitable buffer, even for antibodies that have not been tested in formalin-fixed/paraffin-embedded (FF/PE) sections. Alternatively, you can utilize the Epitope Recovery Buffer Kit to determine the optimal buffer for test antigen unmasking.
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Product Ordering Codes
Types of Epitopes & Recovery Buffers Chart
Epitope Recovery Buffer Selection Chart
Epitope Recovery Optimization Buffer Set
The 2100 Retriever offers the capability to use up to six different buffers simultaneously in one cycle, with each Chamber Slide accommodating one buffer. Each Slide Chamber can hold up to 18 slides, allowing for the optimization of recovery buffers or testing the usability of buffers for epitope recovery for up to 18 individual antibodies in a single run.
Such runs enable the establishment of the best or acceptable epitope recovery conditions for all antibodies, which is particularly crucial when planning multicolor staining of tissue sections for immunofluorescence.
We recommend conducting a primary test for individual antibodies using immunoenzyme staining. This approach facilitates a better analysis of the staining pattern and identification of any potential non-specific staining in certain areas or structures within the section, such as blood vessels.
To simplify the process, we offer a complete set of different recovery buffers for testing purposes (#AP0545-S). Each set contains 125 ml of each 10x buffer stock, providing convenience and facilitating the optimization of recovery conditions.