PrEST Antigens

PrEST Antigens are recombinant protein fragments to use as control antigens

PrEST Antigen™ are recombinant protein fragments used as immunogens for the generation of Triple A Polyclonals™ and PrecisA Monoclonals™. The PrEST Antigens are useful as control antigens, for example as blocking agents and positive assay controls together with the corresponding antibody.

Protein Epitope Signature Tags (PrESTs)

Our primary antibodies are derived from recombinant human Protein Epitope Signature Tags (PrESTs). These protein fragments consist of 50-150 amino acids and are designed to have as low sequence identity as possible to other human proteins.

The PrEST antigens are used for immunization to produce our Triple A Polyclonals and PrecisA Monoclonals. The resulting antibodies are affinity purified against the corresponding PrEST Antigens in a three-step procedure to remove the tag-specific antibodies and to catch the PrEST-specific antibodies. We provide the PrEST Antigens as complements to our antibodies for control experiments.

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Pre-adsorption Assays for IHC

One of the applications for PrEST Antigen is pre-adsorption assays in immunohistochemistry (IHC). The examples below demonstrate IHC staining results following pre-adsorption of antibody with the corresponding PrEST Antigen. The image shows Anti-PLA2R1 (AMAb90772) staining of human kidney, without (left) and with (right) pre-adsorption with PLA2R1 PrEST Antigen (APrEST72110).

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Pre-adsorption Assays for Western Blot

PrEST Antigens are useful for pre-adsorption of the antibody with the corresponding PrEST Antigen in Western blot. The image shows Western blot using the Anti-ALDH1A2 (HPA010022) antibody in human liver lysate, without (left) and with (right) pre-adsorption with the ALDH1A2 PrEST Antigen (APrEST71490).

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Positive Controls in Western Blot

PrEST Antigens can be used as positive controls in Western blot assays. This is exemplified in the image, showing the Anti-AQP4 (HPA014784) antibody in Western Blot using amounts of AQP4 PrEST Antigen (APrEST73067) as loading control ranging from 0.5 to 50 nanograms (right to left).

Designed for Specificity

The protein fragments are expressed as fusion proteins with a dual tag consisting of a hexahistidyl (His6) tag in frame with an immunopotentiating Albumin Binding Protein (ABP)-tag originating from Streptococcal Protein G.

The use of fragments of 50-150 amino acid residues facilitates cloning and protein expression and also provides conformational epitopes that could not be obtained using shorter peptides.

The 50-150 protein specific fragments are selected using a proprietary software to contain unique epitopes present in the native protein suitable for triggering the generation of antibodies of high specificity.

The high specificity is achieved by a complete human genome scanning to ensure that regions with the lowest homology to other human proteins are used as antigens for the generation of antibodies. In addition, signal peptides and membrane regions are avoided in the design.

Production, Validation and Quality Control

For cloning of the PrESTs, a pool of RNA consisting of material from a number of human tissues is used in an RT-PCR approach. The amplified gene fragments are cloned and sequence verified. An E. coli recombinant protein expression system is used for expressing the clones and the products are purified using nickel-containing matrices (IMAC).

The expressed PrEST Antigens are validated using ESI mass spectrometry. Purity is analyzed using SDS page and the PrEST Antigen amount is being quantified using the Nanodrop system.

Blocking Protocol

Get details on how to use PrEST Antigens as control antigens in pre-adsoption assays in IHC, ICC and WB.

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