ANKEF1 Antibody, HRP conjugated

Definition and Composition of ANKEF1 Antibody, HRP Conjugated

ANKEF1 Antibody, HRP conjugated is a rabbit polyclonal antibody specific to the ankyrin repeat and EF-hand domain-containing protein 1 (ANKEF1), covalently linked to horseradish peroxidase (HRP). This conjugate serves as a detection reagent in immunoassays, leveraging HRP’s enzymatic activity for signal amplification in applications like ELISA, Western blotting, and immunohistochemistry.

Primary Uses

• ELISA: Detects ANKEF1 at dilutions of 1:1000–1:5000, depending on conjugation efficiency .

• Western Blotting: Used at 1:100–500 dilutions for protein detection .

• Immunohistochemistry: Effective at 1:10–50 dilutions for tissue staining .

Enhanced Sensitivity via Lyophilization

A modified conjugation method involving lyophilized HRP improves sensitivity by:

1. Reducing reaction volume to increase antibody-HRP collisions.

2. Stabilizing activated HRP for prolonged storage .

Conjugation Protocols

1. Periodate Oxidation:

   • Sodium meta-periodate oxidizes HRP’s carbohydrate groups to aldehydes.

   • Aldehydes react with antibody amines to form Schiff bases, stabilized by sodium cyanoborohydride .

2. Lyophilization:

   • Activated HRP is freeze-dried to concentrate reactive sites, enhancing conjugation efficiency .

Functional Characterization

• UV-Vis Spectrophotometry: Shift in HRP’s 430 nm peak confirms conjugation .

• SDS-PAGE: Conjugates show no migration under reducing conditions, verifying covalent bonds .

• ELISA Standard Curve: Detects ANKEF1 at 1.5 ng with modified conjugates, matching classical methods but requiring lower antibody concentrations .

Cross-Reactivity and Specificity

• No Cross-Reactivity: Affinity-purified antibodies eliminate nonspecific binding .

• Species-Specificity:

  • VWR Product: Mouse-reactive .

  • Abbexa Product: Human-reactive (recombinant immunogen: AA 411–698) .

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