AGO4 Antibody, Biotin conjugated
Description
Structure and Function
Biotin conjugation involves chemically linking biotin (a vitamin with high affinity for streptavidin) to the antibody’s primary or secondary amines. This modification does not interfere with the antibody’s antigen-binding site, preserving specificity while enabling signal amplification in assays like ELISA or Western blot (WB) through streptavidin-horseradish peroxidase (HRP) or alkaline phosphatase (AP) systems .
Western Blotting
Biotin-conjugated AGO4 antibodies are used to detect AGO4 in lysates, often paired with streptavidin-HRP for signal amplification. For example, Agrisera’s AS09 617 (non-conjugated) is validated for WB at 1:2000–1:5000 dilution in Arabidopsis models . Biotin-conjugated variants (e.g., Abbexa’s human AGO4 antibody) enable similar detection with enhanced sensitivity .
Immunoprecipitation (IP)
AGO4 antibodies facilitate IP of RNA-induced silencing complexes (RISCs). In Arabidopsis, floral tissue is recommended due to high AGO4 expression, and proteasome inhibitors like MG132 improve protein stability during extraction .
ELISA
Biotin-conjugated AGO4 antibodies (e.g., ABIN7165100 from antibodies-online) are optimized for ELISA, enabling quantitative detection of AGO4 in human samples .
Biotin Interference in RNA-Ago Interactions
A study by the National Center for Biotechnology Information (NCBI) demonstrated that 3′-biotinylated miRNAs exhibit reduced binding to Ago proteins compared to unmodified miRNAs . While this finding pertains to miRNA biotinylation, it underscores the importance of conjugation site selection in antibody design to avoid steric hindrance. Biotin-conjugated AGO4 antibodies circumvent this issue by attaching biotin to the antibody, not the target RNA or protein .
Specificity and Cross-Reactivity
Agrisera’s AGO4 antibody (AS09 617) is highly specific, showing no cross-reactivity with other Argonaute proteins (e.g., AGO1, AGO2) in Arabidopsis . Similarly, Abbexa’s human AGO4 antibody is purified via Protein G chromatography (>95% purity) to minimize non-specific binding .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Essential for RNA interference (RNAi), AGO4 binds short RNAs, such as microRNAs (miRNAs), repressing translation of complementary mRNAs. It lacks endonuclease activity and does not cleave target mRNAs. AGO4 is also crucial for RNA-directed transcription and replication of the hepatitis delta virus (HDV).
Further research highlights AGO4's multifaceted roles:
- Chimeras with Ago4 N-terminal domains showed defects in RISC activation and, in some cases, RNA loading. This underscores the pleiotropic nature of the Ago N domain and AGO4's unique position within the Ago protein family. PMID: 23748378
- EIF2C4 (AGO4) protein exhibits higher expression in differentiating and terminally differentiated N-type cells compared to S- and I-type cells. PMID: 19393748
