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A reliable, time tested solution to unmask antigen on formalin-fixed sections
Aptum Biologics Ltd.
38 Baker's Drove
Southampton
SO16 8AD
United Kingdom
Epitope Recovery Buffers
Epitopes of different nature may require different recovery buffers for the best results. Most epitopes can be unmasked on FF/PE sections with our R-Universal buffer (considering that the majority of commercial antibodies are prepared against peptide epitope or bacteria-expressed fragment of a protein.)
Nevertheless, we found some cases when buffers, besides R-Universal, allow to achieve a better result. Depending on the structure of the epitope and amino acids comprising it, different buffers should be used for epitope recovery.
The type of epitope defines the most appropriate recovery buffer
As mentioned above, most of currently used commercial antibodies are derived either against a bacterially expressed fragment of target protein or a synthetic peptide epitope. A standard approach used for development of a specific antibody usually means that a well-exposed surface epitope area and/or unique fragment of a protein were chosen for antibody development. However, when an antibody is prepared against a unique mutated or modified epitope, or unique epitope that differs in closely related proteins of the same family of molecules, then the choice of the epitope is rather limited, and it may be not exposed or be a part of a structure which is present only in a mature, folded molecule.
In those cases, R-Universal may be not the best buffer, as well as standard EDTA etc. The chart below may be used to choose the most appropriate buffer for epitope recovery in relation to the type of the 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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In total we offer 5 different buffers for antigen recovery. Most of epitopes can be perfectly recovered in R-Universal buffer, including those usually recovered in Citrate/EDTA/Tris-EDTA buffers. Two new buffers offer solutions for rare, but still existing cases when antibody gives an incorrect pattern on a retrieved section or no staining at all. We also now offer analogues of standard Low (Citrate) and High (EDTA) pH-buffers in case you already use antibodies known to work after tissues being processed in one of these buffers.
Below we provide another chart that helps you to choose the most appropriate buffer even for antibodies that were never tested in FF/PE sections. Alternatively, use the Epitope Recovery Buffer Kit to establish the best buffer in test antigen unmasking.
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Product Ordering Codes
Epitope recovery Optimization Buffer Set
2100 Retriever allows to use up to 6 different buffers in one cycle, each of Chamber slides accommodating one buffer. Each individual Slide Chamber can accommodate up to 18 slides. You can optimize the recovery buffer (and/or test the usability of buffer for recovery) for 18 individual antibodies, in one go.
Such runs allow the establishing of the best/acceptable epitope recovery conditions for all antibodies that can later be used in experiments, which is especially important if you plan an IF multicolour staining of tissue sections.
We advise to do the primary test for individual antibodies with immunoenzyme staining, as this allows for better analyse the pattern of staining and (eventual) non-specific staining of some areas/structures in section (i.e. blood vessels etc.)
For your convenience, we offer a complete set of different recovery buffers for the tests (#AP0545-S), containing 125 ml of each 10x buffer stock.
Types of Epitopes & Recovery Buffers Chart
Epitope Recovery Buffer Selection Chart