The identification of high-performing antibodies for RNA-binding protein FUS for use in Western Blot, immunoprecipitation, and immunofluorescence - PubMed (original) (raw)

The identification of high-performing antibodies for RNA-binding protein FUS for use in Western Blot, immunoprecipitation, and immunofluorescence

Walaa Alshalfie et al. F1000Res. 2023.

Abstract

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community. In this study, we characterized ten FUS commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Keywords: FUS; RNA-binding protein FUS; Uniprot ID P35637; Western Blot; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation.

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Conflict of interest statement

Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABclonal -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems -Thermo Fisher Scientific.

Figures

Figure 1.. FUS antibody screening by Western Blot.

Lysates of HeLa (WT and_FUS_ KO) were prepared and 30 μg of protein were processed for Western Blot with the indicated FUS antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. An exception was given for antibody GTX101810, which was titrated to 1/3000, as the signal was too weak when following the supplier’s recommendation. Antibody dilution used: NBP2-52874* at 1/1000; GTX101810 at 1/3000; GTX01039* at 1/1000; 60160-1-Ig* at 1/10000; 11570-1-AP at 1/4000; MA3-089* at 1/2000; MA5-32483** at 1/1000, ab124923** at 1/5000; ab154141* at 1/1000; ab243880** at 1/1000. Predicted band size: 53 kDa. Observed specific band size: ~70 kDa. *Monoclonal antibody; **Recombinant antibody.

Figure 2.. FUS antibody screening by immunoprecipitation.

HeLa lysates were prepared, and IP was performed using 1.0 μg of the indicated FUS antibodies pre-coupled to protein G or protein A Sepharose beads. Samples were washed and processed for Western Blot with the indicated FUS antibody. For Western Blot, NBP2-52874* and ab243880** were used at a dilution of 1/2000. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitated. *Monoclonal antibody; **Recombinant antibody.

Figure 3.. FUS antibody screening by immunofluorescence.

HeLa WT and_FUS_ KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio on coverslips. Cells were stained with the indicated FUS antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody. Acquisition of the green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative images of the red (grayscale) channels are shown. WT and KO cells are outlined with yellow and magenta dashed line, respectively. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies GTX101810, GTX01039*, 60160-1-Ig*, 11570-1-AP, MA5-32483** and ab124923**, which were titrated as the signals were too weak when following the supplier’s recommendations. Antibody dilution used: NBP2-52874* at 1/1000; GTX101810 at 1/700; GTX01039* at 1/1000; 60160-1-Ig* at 1/2000; 11570-1-AP at 1/1000; MA3-089* at 1/1000; MA5-32483** at 1/1000; ab124923** at 1/1000; ab154141* at 1/1000; ab243880** at 1/500. Bars=10 μm. *Monoclonal antibody; **Recombinant antibody.

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The identification of high-performing antibodies for RNA-binding protein FUS for use in Western Blot, immunoprecipitation, and immunofluorescence - PubMed (original) (raw)