# Fluorescence‐Based Multiplex Western Blot to Simultaneously Detect the Insulin‐Like Growth Factor‐1 (IGF‐1) Isoforms

## TL;DR

A new fluorescence-based method allows the simultaneous detection of different IGF-1 protein forms, helping to study their roles in growth and development.

## Contribution

A novel multiplex Western blot system using dual-epitope detection for IGF-1 isoforms is introduced.

## Key findings

- A dual-antibody fluorescence system successfully detects mature IGF-1, proIGF-1s, and E-peptides in the same sample.
- The method distinguishes IGF-1 isoforms using antibodies targeting different epitopes, increasing detection specificity.
- This approach is feasible for studying IGF-1 isoform functions in biological processes.

## Abstract

Insulin‐like growth factor‐1 (IGF‐1) is critical for tissue growth and development. The IGF‐1 gene contains six exons and due to alternative splicing three different isoforms might be produced: the IGF‐1Ea, Eb, and Ec prohormones (proIGF‐1s). These proIGF‐1s share the same IGF‐1 mature sequence, which is responsible for the IGF‐1 receptor binding but differ in their carboxy‐terminal extensions called Ea‐, Eb‐, and Ec‐peptides. Several lines of evidence indicate that E‐peptides control the intracellular proIGF‐1s localization and maturation. Here, we present a multiplex Western blotting system able to simultaneously discriminate and quantify mature IGF‐1, proIGF‐1s and E‐peptides within the same sample. HEK293 cells were transiently transfected with plasmids containing the IGF‐1Ea, IGF‐1Eb, or IGF‐1Ec isoform or an empty vector. Two different primary antibodies, which recognize the mature sequence or the common region of E‐peptides, were used to detect IGF‐1 isoforms, which were subsequently distinguished with secondary antibodies conjugated to different fluorophores. Our results demonstrate the feasibility of simultaneously detecting different IGF‐1 isoforms using two primary antibodies directed against different epitopes of proIGF‐1s, combined with fluorescence‐conjugated secondary antibodies. Furthermore, this dual‐epitope strategy increases the specificity of protein detection, making it a valuable tool for studying the diverse roles of IGF‐1 isoforms in biological processes.