Davide De Forni, Barbara Poddesu, Giulia Cugia, James Chafouleas, Julianna Lisziewicz, Franco Lori.
Abstract
ELISA or Western blot is known as a basic technique to be used for measurement of intracellular proteins, but in some cases, they cannot overcome problems such as normalization between samples or extraneous costs for required commercial kits. In order to address this problem, we developed a rapid and effective method (a hybrid of Western blot and ELISA). We use this new hybrid method to detect and normalize trace protein changes in gene expression intracellularly at a lower cost.
Introduction
It is known that ELISA (enzyme-linked immunosorbent assay) is an assay technique used to measure proteins, which is commercially available in various different kits. Utilization of the correct corresponding ELISA kit is a more convenient and fast method to measure/determine trace amounts of proteins without using many facilities and equipment. However, we can occasionally encounter difficulties such as the normalization of the relative protein levels among multiple test groups. Additionally, the cost of the commercial ELISA kit is very expensive.
Materials and Methods:
Mouse RAW264.7 macrophage-like cells (Cat# TIB-71™, ATCC®) or human THP-1 monocyte-like cells (Cat# TIB-202™, ATCC®) were grown in RPMI medium 1640 (Cat#: 11875–093, Life Technologies, NY) with 10% FBS (Fetal Bovine Serum, Sigma) at 37°C in a 5% CO2 atmosphere.
Discussion
A hybrid of Western blot with ELISA has been recently reported to quantify the accuracy of protein in 96-well microplate for some experiments such as In-situ Protein Detection or In-cell Western (ICW). However, this hybrid technique has only been applied to cells cultured on 96-well microplates and its reproducibility of the data can be greatly affected by intermediate steps or uncontrolled factors, such as protein sample preparation, protein fixation, normalization, or false positives.
Citation: Edabashi H, Elghadafi R, Rajkanth N, Mody JS, Ma W, Dibart S, et al. (2023) A hybrid technique for measurement of intra/extracellular proteins. PLoS ONE 18(5): e0282948. https://doi.org/10.1371/journal.pone.0282948
Editor: Zhaoqing Luo, Purdue University, UNITED STATES
Received: August 9, 2022; Accepted: February 27, 2023; Published: May 4, 2023.
Copyright: © 2023 Edabashi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
