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Data Note

The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence

[version 1; peer review: 2 approved with reservations]
PUBLISHED 13 Feb 2023
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This article is included in the YCharOS (Antibody Characterization through Open Science) gateway.

Abstract

Moesin is a cytoskeletal adaptor protein, involved in the modification of the actin cytoskeleton, with relevance to Alzheimer’s Disease. Well characterized anti-Moesin antibodies would benefit the scientific community. In this study, we characterized ten commercial antibodies for Moesin in 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 well-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Keywords

Uniprot ID P26038, MSN, Moesin, antibody characterization, antibody validation, Western Blot, immunoprecipitation, immunofluorescence

Introduction

Moesin is a cytoskeletal adaptor protein that belongs to the Ezrin-Radixin-Moesin family of proteins that connect the actin cytoskeleton to the plasma membrane, regulating the structure and function of specific domains of the cell cortex.1,2 Moesin plays a pertinent role in immunity, acting on T and B-cell homeostasis and self-tolerance.3,4

Proteomic and protein co-expression network analysis of Alzheimer's Disease (AD) brain has revealed a module that is enriched in inflammation-related proteins.5 Moesin, along with CD44 antigen, have emerged as key drivers in this inflammation module. Disrupting the Moesin-CD44 pathway is a current focus in AD research.6 Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.

Here, we compared the performance of a range of commercially available antibodies for Moesin and identified high-performing antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of Moesin properties and function.

Results and discussion

Our standard protocol involves comparing readouts from wild-type and knockout cells.7,8 The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log2 (transcripts per million “TPM” +1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available HeLa cells expressed the Moesin transcript at RNA levels above the average range of cancer cells analyzed. Parental and MSN knockout HeLa cells were obtained from Abcam (Table 1).

Table 1. Summary of the cell lines used.

InstitutionCatalog numberRRID (Cellosaurus)Cell linegenotype
Abcamab255448CVCL_0030HeLaWT
Abcamab265020CVCL_B9VNHeLaMSN KO

For Western Blot, we resolved proteins from wild-type and MSN KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1).

3815c5d8-ce67-4027-a759-ba7c874ef0ed_figure1.gif

Figure 1. Moesin antibody screening by Western Blot.

Lysates of HeLa (WT and MSN KO) were prepared, and 25 μg of protein were processed for Western Blot with the indicated Moesin 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 based on the recommendations provided by suppliers with exceptions for antibodies MA5-32231**, MA5-17130* and GTX101708, which were titrated as the signal received was too strong following the supplier’s recommendations. The antibody dilutions were as follows: MA5-32231** at 1/5000, MA5-17130* at 1/5000, NBP2-32876* at 1/5000, NBP2-44579* at 1/5000, NBP2-44580* at 1/5000, GTX101708 at 1/5000, ab52490** at 1/1000, ab151542** at 1/1000, ab169789** at 1/10000 and ab193380** at 1/400. Predicted band size: 68 kDa. *Monoclonal antibody, **Recombinant antibody.

For immunoprecipitation, we used the antibodies to immunopurify Moesin from HeLa cell extracts. The performance of each antibody was evaluated by detecting the Moesin protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).

3815c5d8-ce67-4027-a759-ba7c874ef0ed_figure2.gif

Figure 2. Moesin antibody screening by immunoprecipitation.

HeLa lysates were prepared, and IP was performed using 1.0 μg of the indicated Moesin antibodies pre-coupled to either protein G or protein A Sepharose beads. Samples were washed and processed for Western Blot with the indicated Moesin antibody. For immunoblot, NBP2-44579*, GTX101708 and ab169789** were used at 1/20000, 1/20000 and 1/10000, respectively. 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 = immunoprecipitate. *Monoclonal antibody, **Recombinant antibody.

For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy.9 In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce imaging and analysis bias (Figure 3).

3815c5d8-ce67-4027-a759-ba7c874ef0ed_figure3.gif

Figure 3. Moesin antibody screening by immunofluorescence.

HeLa WT and MSN 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 Moesin antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with yellow and magenta dashed line, respectively. Antibody dilutions were chosen based on supplier recommendations, except for MA5-32231** which was titrated to 1/1000 as the signal was too strong. When concentrations were not provided by the supplier, antibodies were tested at 1/1000, which was the case for MA5-17130*. Antibody dilutions used; MA5-32231** at 1/1000; MA5-17130* at 1/1000; NBP2-32876* at 1/200; NBP2-44579* at 1/200; NBP2-44580* at 1/200; GTX101708 at 1/200; ab52490** at 1/200, ab151542** at 1/200, ab169789** at 1/100, ab193380** at 1/200. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody.

In conclusion, we have screened Moesin commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence, and identified several high-quality antibodies under our standardized experimental conditions.

Methods

Antibodies

All Moesin antibodies are listed in Table 2. Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424).

Table 2. Summary of Moesin antibodies tested.

CompanyCatalog numberLot numberRRID (Antibody Registry)ClonalityClone IDHostConcentration (μg/μl)Vendors recommended applications
Thermo Fisher ScientificMA5-32231**VJ3101165AB_2809517recombinant-monoSC69-01rabbit1.00Wb, IF
Thermo Fisher ScientificMA5-17130*VJ3101185AB_2538601monoclonal2C12mouse1.00Wb
Bio-TechneNBP2-32876*4478-1XP160531AB_2885048monoclonalSPM562mouse0.20Wb, IF
Bio-TechneNBP2-44579*44578-2P190315AB_2885047monoclonalMSN/492mouse0.20Wb, IF
Bio-TechneNBP2-44580*4478-3P190605AB_2885046monoclonalMSN/493mouse0.20Wb, IF
GeneTexGTX10170840198AB_10618789polyclonal-rabbit0.21Wb, IP, IF
Abcamab52490**GR3207377-11AB_881245recombinant-monoEP1863Yrabbit0.20Wb, IP, IF
Abcamab151542**GR112662-8AB_2893185recombinant-monoEPR2428(2)rabbit0.09Wb, IF
Abcamab169789**GR121830-3AB_2885098recombinant-monoEPR2429(2)rabbit0.07Wb, IP, IF
Abcamab193380*GR3373113-1AB_2885109monoclonalMSN/491mouse0.20Wb, IP

Cell culture

HeLa WT and MSN KO cells used are listed in Table 1. Cells were cultured in DMEM high-glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (GE Healthcare cat. Number SH30072.03), 2 mM L-glutamate (Wisent cat. number 609065, 100 IU penicillin and 100 μg/ml streptomycin (Wisent cat. number 450201).

Antibody screening by Western Blot

Western Blots were performed as described in our standard operating procedure.10 HeLa WT and MSN KO cells were collected in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1.0 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor. Lysates were sonicated briefly and incubated 30 min on ice. Lysates were spun at ~110,000 × g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western Blot.

Western Blots were performed with large 4–15% gradient polyacrylamide gels and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show together with individual Western Blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated O/N at 4°C with 5% bovine serum albumin in TBS with 0.1% Tween 20 (TBST). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/ml in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes are incubated with ECL from Pierce (cat. number 32106) prior to detection with HyBlot CL autoradiography films from Denville (cat. number 1159T41).

Antibody screening by immunoprecipitation

Immunoprecipitation was performed as described in our standard operating procedure.11 Antibody-bead conjugates were prepared by adding 1.0 μg of antibody to 500 μl of PBS with 0.01% triton X-100 in a microcentrifuge tube, together with 30 μl of protein A - (for rabbit antibodies) or protein G - (for mouse antibodies) Sepharose beads. Tubes were rocked overnight at 4°C followed by two washes to remove unbound antibodies.

HeLa cells were collected in HEPES buffer (20 mM HEPES, 100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor. Lysates are rocked 30 min at 4°C and spun at 110,000 × g for 15 min at 4°C. One ml aliquots at 1 mg/ml of lysate were incubated with an antibody-bead conjugate for ~2 hrs at 4°C. Following centrifugation, the unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of HEPES lysis buffer and processed for SDS-PAGE and Western Blot on a 4-15% acrylamide gel.

Antibody screening by immunofluorescence

Immunofluorescence was performed as described in our standard operating procedure.9 HeLa cells (WT and MSN KO) were labelled with a green dye and with a deep red fluorescent dye from Abcam (cat. number ab176735 and ab176736), respectively. WT and KO cells were plated on glass coverslips as a mosaic and incubated for 24 hrs in a cell culture incubator. Cells were fixed in 4% PFA (in PBS) for 15 min at room temperature and then washed 3 times with PBS. Cells were permeabilized in PBS with 0.1% Triton X-100 for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum and 0.01% Triton X-100 for 30 min at room temperature. Coverslips were incubated face down on a 50 μl drop (on paraffin film in a moist chamber) with IF buffer (PBS, 5% BSA, 0,01% Triton X-100) containing the primary Moesin antibodies O/N at 4°C. Cells were washed 3 times for 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies, including DAPI, in IF buffer at a dilution of 1.0 μg/ml for 1 hr at room temperature. Cells were washed 3 times for 10 min with IF buffer and once with PBS. Coverslips were mounted on a microscopic slide using fluorescence mounting media (DAKO).

Imaging was performed using a Zeiss LSM 880 laser scanning confocal microscope equipped with a Plan-Apo 40× oil objective (NA = 1.40). Analysis was done using Image J. All cell images represent a single focal plane. Figures were prepared using Adobe Photoshop to adjust contrast, apply 1 pixel Gaussian blur and then assembled with Adobe Illustrator.

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Alshafie W, Ayoubi R, Fotouhi M et al. The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved with reservations]. F1000Research 2023, 12:172 (https://doi.org/10.12688/f1000research.130126.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
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PUBLISHED 13 Feb 2023
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Reviewer Report 02 Aug 2023
Sungsoo Na, Indiana University Purdue University Indianapolis, Indianapolis, Indiana, USA 
Approved with Reservations
VIEWS 27
This manuscript compared the commercially available antibodies through various assays, including Western blot, immunoprecipation, and immunofluorescence. All the experimental methods and data are well presented. However, this manuscript lacks descriptions on the comparison, characterization, and identification of the antibodies in ... Continue reading
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HOW TO CITE THIS REPORT
Na S. Reviewer Report For: The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved with reservations]. F1000Research 2023, 12:172 (https://doi.org/10.5256/f1000research.142858.r177143)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 29 Nov 2023
    Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada
    29 Nov 2023
    Author Response
    Thank you Sungsoo Na for reviewing this article.

    As a third-party entity, YCharOS refers to remain unbiased which is why the authors do not compare the performance of antibodies ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 29 Nov 2023
    Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada
    29 Nov 2023
    Author Response
    Thank you Sungsoo Na for reviewing this article.

    As a third-party entity, YCharOS refers to remain unbiased which is why the authors do not compare the performance of antibodies ... Continue reading
Views
27
Cite
Reviewer Report 02 Aug 2023
Lyndsay Avery, Biology, Saint Michael's College, Colchester, Vermont, UK 
Approved with Reservations
VIEWS 27
In this manuscript, the authors aim to validate 10 commercially available moesin antibodies for western blot, immunoprecipitation, and immunofluorescence. This work makes evident that there is great variability in both what each antibody is suited for and the conditions in ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Avery L. Reviewer Report For: The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved with reservations]. F1000Research 2023, 12:172 (https://doi.org/10.5256/f1000research.142858.r177135)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 29 Nov 2023
    Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada
    29 Nov 2023
    Author Response
    Thank you, Lyndsay Avery, for your response, your feedback is much appreciated.

    We will be submitting a new version of the manuscript with your following suggestions included. In the ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 29 Nov 2023
    Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada
    29 Nov 2023
    Author Response
    Thank you, Lyndsay Avery, for your response, your feedback is much appreciated.

    We will be submitting a new version of the manuscript with your following suggestions included. In the ... Continue reading

Comments on this article Comments (0)

Version 3
VERSION 3 PUBLISHED 13 Feb 2023
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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