Page 95 - GPD-2-2

Gene & Protein in Disease

SHORT COMMUNICATION
How the West(ern) was won: Solutions for

immunoblotting large and small proteins

1
2
Paula Llabata * and Pere Llinàs-Arias *
1 Cancer Genetics Group, Josep Carreras Leukaemia Research Institute, 08916 Barcelona, Spain
2 Cancer Epigenetics Laboratory, Cancer Cell Biology Group, Institut d’Investigació Sanitària Illes
Balears (IdISBa), 07122 Palma, Spain

Abstract

Relative protein quantification is a well-established technique in the vast majority
of molecular biology laboratories. However, western blot standard protocols
may not detect proteins of certain sizes. When the molecular weight of protein of
interest is out of the 10–250 kDa range, its migration through the gel or transfer to
the membrane is compromised, making its detection difficult. Here, we present a
set of modifications of the standard working procedure for western blotting based
on the experience working with small VCP-interacting protein and MAX-gene-
associated protein, whose molecular weights are 8 and 350 kDa, respectively. We
expect that these adaptations may help researchers to improve their experiments in
a cost-effective manner.

Keywords: Western blot; Immunoblotting; Large proteins; Small proteins;
Small VCP-interacting protein; MAX-gene-associated protein
*Corresponding authors:
Pere Llinàs-Arias
(pere.llinas@ssib.es)
Paula Llabata
(paulallabata@gmail.com) 1. Introduction
Citation: Llabata P, Llinàs-Arias P, In this century, the rise of high throughput techniques has allowed an unprecedented
2023, How the West(ern) won: advance in our knowledge about molecular biology [1,2] . The implementation of “omics”
Solutions for immunoblotting large
and small proteins. Gene Protein Dis, (including genomics, proteomics, transcriptomics, and metabolomics, among others)
2(2):0547. and their combination has contributed to a better understanding of different diseases by
https://doi.org/10.36922/gpd.0547 describing new biological networks, connecting the dots, and finding out their clinical
Received: April 18, 2023 relevance . Although the development of these techniques is encouraging, the results
[3]
Accepted: June 15, 2023
Published Online: June 30, 2023 derived from these studies must be validated through different classic methods. In
addition, many studies focused on a single pathway, where a simpler and cost-effective
Copyright: © 2023 Author(s).
This is an Open-Access article method is needed. Regarding the study of proteins, the standard method is Western blot.
distributed under the terms of the Western blot is a well-established technique that is performed in the vast majority of
Creative Commons Attribution
License, permitting distribution, and molecular biology laboratories to determine the relative protein abundance in different
reproduction in any medium, which experimental conditions. A standard protocol usually works for a large proportion of
provided that the original work is
properly cited. proteins between 10 and 250 kDa (Figure 1A) after antibody optimization. However,
these procedures are invalid when the target protein size is not in the above-mentioned
Publisher’s Note: AccScience
Publishing remains neutral with range.
regard to jurisdictional claims in
published maps and institutional Here, we describe a set of modifications from our laboratories’ standard working
affiliations. procedure (SWP) for performing western blots to detect small or large proteins.

Volume 2 Issue 2 (2023) 1 https://doi.org/10.36922/gpd.0547