# Cross-Reactivity of N6AMT1 Antibodies with Aurora Kinase A: An Example of Antibody-Specific Non-Specificity

## TL;DR

This paper shows that some antibodies meant for N6AMT1 also mistakenly bind to Aurora kinase A, highlighting the need for careful antibody validation in research.

## Contribution

The study reveals a specific cross-reactivity mechanism between N6AMT1 antibodies and Aurora kinase A due to a shared motif.

## Key findings

- Three commercial N6AMT1 antibodies cross-react with Aurora kinase A.
- The cross-reactivity is due to a shared ENNPEE motif in both proteins.
- This highlights the importance of controls in antibody-based experiments.

## Abstract

Primary antibodies are one of the main tools used in molecular biology research. However, the often-occurring cross-reactivity of primary antibodies complicates accurate data analysis. Our results show that three commercial polyclonal antibodies raised against N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) strongly cross-react with endogenous and recombinant mitosis-associated protein Aurora kinase A (AURKA). The cross-reactivity was verified through immunofluorescence, immunoblot, and immunoprecipitation assays combined with mass spectrometry. N6AMT1 and AURKA are evolutionarily conserved proteins that are vital for cellular processes. Both proteins share the motif ENNPEE, which is unique to only these two proteins. We suggest that N6AMT1 antibodies recognise this motif in N6AMT1 and AURKA proteins and exhibit an example of “specific” non-specificity. This serves as an example of the importance of controls and critical data interpretation in molecular biology research.