PrEST Antigens - recombinant protein fragments to use as control antigens

PrEST Antigen™ are recombinant protein fragments used as immunogens to generate 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 catch the PrEST-specific antibodies. We provide the PrEST Antigens as complements to our antibodies for control experiments.

Pre-adsorption Assays for IHC

One of the applications for PrEST Antigen is pre-adsorption assays in immunohistochemistry (IHC). The example on the picture shows the IHC staining of the human kidney using the Anti-PLA2R1 (AMAb90772) monoclonal antibody without (left) and with (right) pre-adsorption with the correspondent PLA2R1 PrEST Antigen (APrEST72110). 

See Protocol

Pre-adsorption Assays for Western Blot

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

See Protocol

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 the 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 immuno-potentiating 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 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 the PrESTs, a pool of RNA consisting of material from several human tissues is used in an RT-PCR approach. First, the amplified gene fragments are cloned and the sequence verified. Then, 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 the SDS page, and the PrEST Antigen amount is quantified using a Nanodrop system.