A guide to selecting high-performing antibodies for Secreted frizzled-related protein 1 (sFRP-1) for use in Western Blot and immunoprecipitation

Secreted frizzled-related protein 1 (sFRP-1) is a secreted protein, belonging to the secreted glycoprotein SFRP family. As a modulator of the Wnt/β-catenin signalling pathway, sFRP-1 has implications in human cancers and neurological diseases. If the community had access to well-characterized anti-sFRP-1 antibodies, the reproducibility of sFRP-1 research would be enhanced. In this study, we characterized 11 sFRP-1 commercial antibodies for Western Blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address the antibody reproducibility issue by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Introduction SFRP-1 is a secreted protein belonging to the secreted frizzled-related protein (SFRP) family. 1 Having an amino acid terminal cysteine-rich domain homologous to the putative Wnt-binding domain of frizzled receptors, sFRP-1 functions as a modulator of the Wnt/β-catenin signalling pathway that is essential to multiple cellular processes. 2,3sregulated Wnt/β-catenin activity levels play a role in various disease processes. 4Epigenetic silencing of SFRP1 elevates Wnt/β-catenin activity, which has been linked to cancer. 2,44][5] Targeting sFRP-1 to restore the Wnt/β-catenin signalling pathway is of current interest in AD research to discover novel therapies. 3Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.
7][8] Here, we compared the performance of a range of commercially available antibodies for sFRP-1 use in Western Blot and immunoprecipitation.This article serves as a valuable guide to help researchers select high-quality antibodies for their specific needs, facilitating the biochemical and cellular assessment of sFRP-1 properties and function.

Results and discussion
Our standard protocol involves comparing readouts from wild-type and knockout (KO) cells. 9,10The 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 log 2 (transcripts per million "TPM" +1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655).Commercially available A549 cells expressed the sFRP-1 transcript at RNA levels above the average range of cancer cells analyzed.Parental and SFRP1 knockout A549 cells were obtained from Abcam (Table 1).SFRP-1 is predicted to be a secreted protein.Accordingly, we collected concentrated culture media from both wild-type and SFRP1 KO cells and used the conditioned media to probe the performance of the antibodies (Table 2) side-by-side by Western blot and immunoprecipitation.The profiles of each of the antibodies are shown in Figures 1 and 2. The datasets can be found as Underlying data. 13,14 conclusion, we have screened eleven sFRP-1 commercial antibodies by Western Blot and immunoprecipitation.Several high-performing antibodies that successfully detect sFRP-1 under our standardized experimental conditions.In our effort to address the antibody reliability and reproducibility challenges in scientific research, the authors recommend the antibodies that demonstrated to be underperforming under our standard procedure be removed from the commercial antibody market.However, the authors do not engage in result analysis or offer explicit antibody recommendations.A limitation of this study is the use of universal protocolsany conclusions remain relevant within the confines of the experimental setup and cell line used in this study.Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles.This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs.

Antibodies
All sFRP-1 antibodies are listed in Table 2. Peroxidase-conjugated goat anti-rabbit and donkey anti-goat antibodies are from Thermo Fisher Scientific (cat.number 65-6120 and A15999).Antibody screening by Western Blot on culture media A549 cells (WT and SFRP1 KO) were washed 3Â with PBS and starved for ~18 hrs.Culture media were collected and centrifuged for 10 min at 500 Â g to eliminate cells and larger contaminants, then for 10 min at 4500 Â g to eliminate smaller contaminants.Culture media were concentrated by centrifuging at 4000 Â g for 10 min using Amicon Ultra-15 Centrifugal Filter Units with a membrane NMWL of 10 kDa (MilliporeSigma cat.number UFC901024).

Cell
Western Blots were performed as described in our standard operating procedure. 11Western Blots were performed with large 8-16% 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 overnight 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 more washes with TBST.Membranes were 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 on culture media Immunoprecipitation was performed as described in our standard operating procedure. 12Antibody-bead conjugates were prepared by adding 1 μg of antibody to 500 ul of Pierce IP Buffer from Thermo Fisher Scientific (cat.number 87788) in a microcentrifuge tube, together with 30 μl of Dynabeads protein A -(for rabbit antibodies) or protein G -(for goat antibodies) from Thermo Fisher Scientific (cat.number 10002D and 10004D, respectively).Pierce IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) was supplemented with the Halt Protease Inhibitor Cocktail 100X from Thermo Fisher Scientific (cat.number 78446) at a final concentration of 1Â.Tubes were rocked for ~2 hrs at 4°C followed by two washes to remove unbound antibodies.
Starved A549 WT culture media were concentrated as described above. 1 ml aliquots at 0.5 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 IP Lysis Buffer and processed for SDS-PAGE and Western Blot on 8-16% polyacrylamide gels.Prot-A: HRP (MilliporeSigma, cat.number P8651) was used as a secondary detection system at a dilution of 0.4 μg/ml.

Lynne Howells
University of Leicester, Leicester, UK The article describes a protocol that will facilitate the use of better antibody tools in the detection of the sFRP-1 protein via western blot and IP techniques.
The article is succinct, well written and will provide an enabler for researchers looking to analyse this specific protein.
Even though the article is short, there are a number of areas where 'lab slang' has crept in, which should be rectified.: When saying cells were starved -specify serum starved, or say that media was subsequently replaced with serum-free media.
In the Ab screening by IP section -it says 500 uL instead of micro (mu symbol) Millilitres should be mL rather than ml

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
The authors have not addressed any of my concerns.However, I note that they have made a qualifying statement in their report that "any conclusions drawn remain relevant within the confines of the experimental set up and cell line used in this study."I accept that those in the field will be able to interpret the available data in the light of their expertise.
As such, I understand that the authors need only to provide information on why and how the data were created but do not need to include analyses or conclusions.Indeed, the authors did not address any of my concerns, some of which requires additional experiments or an explanation of seemingly contradictory data between Figure 1 and 2. Since the instructions to authors is that analyses or conclusions are not required, I am not sure if I am asking more than is required of the authors.It would be great if the authors could at least provide a point-by-point rebuttal of my concerns in the comments section.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Biomarkers, antibody generation/characterization and validation, antigen design, recombinant protein expression, genetic engineering, assay development.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
concentrated culture media collected from starved A549 cells by western blotting.However, these antibodies were able to detect sFRP-1 at ~35 kDa position in the starting material samples in Figure 2 which are essentially equivalent to the WT samples in Figure 1.
In Figure 2, as indicated in the legend, all starting material and immunoprecipitates corresponding to each antibody tested are western blotted with one antibody, MA5-38193.Since MA5-38193 detects sFRP-1 by WB (Figure 1), signal is visible in all SM lanes.
The detailed antibody characterization platform we have created, along with academic and industry researchers, is available on Protocol Exchange.https://doi.org/10.21203/rs.3.pex-2607/v1transcript levels.However, high transcript levels do not necessarily translate to high protein expression.The A549 and A549 sFRP1 KO cell lines as well as some of the antibodies tested were from the same vendor.To have added confidence that the selected antibodies are recognizing endogenous sFRP-1 expressed in A549 cells, the authors should include a recombinant source of sFRP-1 from a different vendor in their western blots.In others words, each antibody is tested against endogenous sFRP-1 expressed in A549 cells, A549 SFRP1 KO (negative control) and recombinant sFRP-1 (eg R&D Systems cat no.1384-SF).

2.
Human sFRP-1 is a member of the SFRP family.Since many of the antibodies selected are polyclonal in nature, the authors should assess potential cross-reactivity against other closely related family members eg human sFRP-2, sFRP-4 and sFRP-5.Sequence alignment between sFRP-1 against these family members shows stretches of 100% identical amino acids up to 22 amino acids long, portending potential cross-reactivity of anti-sFRP-1 polyclonal antibodies against these entities.Cross-reactivity of the recombinant monoclonal antibodies should also be tested since there is no epitope information for these antibodies.

3.
The results for GTX4193, GTX102371, AF1384, AB4193 and AB84003 in Figure 1 and 2 appear contradictory.In Figure 1, these antibodies could not detect sFRP-1 present in concentrated culture media collected from starved A549 cells by western blotting.However, these antibodies were able to detect sFRP-1 at ~35 kDa position in the starting material samples in Figure 2 which are essentially equivalent to the WT samples in Figure 1.

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Biomarkers, antibody generation/characterization and validation, antigen design, recombinant protein expression, genetic engineering, assay development.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
good for the scientific community.That being said, we understand how this intention may be misinterpreted.A new version has been submitted, to include modifications to the title, introduction and results&discussion section that ensure our goals clearly are aligned and defined to all viewers.
References 8-11 have been edited and will be complete in the newly submitted version.
Thank you for your feedback and corrections!Competing Interests: No competing interests were disclosed.
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Table 1 .
Summary of the cell lines used.In this new version of the article, statements have been added to the Abstract, Introduction and Results & Discussion sections to ensure the YCharOS initiative and goals are clearly defined to prevent misinterpretation.

Table 2 .
Summary of the Secreted frizzled-related protein 1 antibodies tested.