PrEST Antigens

Atlas Antibodies has introduced a new product family, PrEST Antigens. PrEST Antigens are recombinant protein fragments used as immunogens for the generation of Triple A Polyclonals and PrecisA Monoclonals. PrEST Antigens are available for use as blocking agents and positive assay controls together with the corresponding antibody.

PrEST Antigens to Complement Atlas Antibodies

The antibodies in Atlas Antibodies' catalog 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. The resulting polyclonal 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. Atlas provide the PrEST Antigens as complements to their antibodies for control experiments.

PrEST Antigens in Pre-adsorption Assays for Immunohistochemistry

One of the applications for PrEST Antigen is pre-adsorption assays in immunohistochemistry (IHC). The examples below demonstrates IHC staining results following pre-adsorption of antibody with the corresponding PrEST Antigen.


 
Anti-PLA2R1, AMAb90772 Staining of human kidney, with and without preadsoption with PLA2R1 PrEST Antigen
 Anti-SOX11, HPA000536 Staining of human mantle cell lymphoma, with and without preadsoption with SOX11 PrEST Antigen

Protocol

PrEST Antigens in Pre-adsorption Assays for Western Blot

PrEST Antigens are useful for pre-adsorption of antibody with the corresponding PrEST Antigen in Western Blot.

Anti-STAT3, HPA001671 Western Blot using Anti-STAT3 antibody in human cell line RT-4, with and without pre-adsorption with STAT3 PrEST Antigen.

Protocol

PrEST Antigens as Positive Controls in Western Blot

PrEST Antigens can be used as positive controls controls in Western Blot assays.

Anti-ALDH1A2, HPA010022 Western Blot using amounts of ALDH1A2 PrEST Antigen as loading control ranging from 0.5 to 50 nano grams.

Designed for Specificiy

The protein fragments are expressed as fusion proteins with a dual tag consisting of a hexahistidyl (His6) tag in frame with an immunopotentiating Albumin BinĀ­ding 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.

PrEST Antigen Production

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).

PrEST Antigen Validation and Quality Control

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.


Related to:
Brands: Atlas Antibodies
Product groups: Proteins / Signaling Molecules