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

Bocconcelli M, Fanelli F, Saltarelli R, De Santi M, Barone R, Barbieri E, Annibalini G

Electrophoresis19 Mar 2025

PMID40105249PMCIDPMC12039166DOI10.1002/elps.8116PepBankPUB-40105249

Summary

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.

CC BYText from Europe PMC. Highlighted sentences are the source of results.

Results from this study1

No result from this study comes from a test of the peptide.

Studies that did not test the peptide alone1

In this study, MGF / PEG-MGF was a lab tool or a carrier, part of a mix, or the subject of a lab method. These results are not evidence about taking MGF / PEG-MGF.

01LabNot a test of MGF / PEG-MGF aloneIn vitro (cells)Other
In HEK293 cells transfected with IGF-1Ea, Eb or Ec plasmids, a dual-epitope multiplex Western blot detected the IGF-1 isoforms at the same time.
In plain wordsIn lab tests, a new blotting method detected different IGF-1 forms in one sample.
Peptide
MGF / PEG-MGF
Outcome
Not applicableSimultaneous detection of IGF-1 isoforms by multiplex Western blot
Study
humann: not reported in source
HEK293 cells transfected with IGF-1 isoform plasmids
Outcome group
Receptor activity and mechanism · Pharmacology
Source sentences
Summary
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.
1 context sentence(design, population, supporting numbers)
Summary · context
HEK293 cells were transiently transfected with plasmids containing the IGF-1Ea, IGF-1Eb, or IGF-1Ec isoform or an empty vector.
In this study, MGF / PEG-MGF was a lab tool or a carrier, part of a mix, or the subject of a lab method. This result is not evidence about taking MGF / PEG-MGF.
Show in the study text Report a problemFND-4010524901

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Version history

PepBank keeps every version of the data on this study. Status changes from Retraction Watch, Europe PMC and Crossref are listed with their source.

Version 2Current
13 Oct 2026, 21:44 UTC
Study results updated
Version 1
13 Oct 2026, 16:31 UTC
First added · source update from Europe PMC

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