Identification of high-performing antibodies for SPARC-related modular calcium-binding protein 1 (SMOC-1) for use in Western Blot and immunoprecipitation

SPARC-related modular calcium-binding protein 1, otherwise known as SMOC-1, is a secreted glycoprotein involved in various cell biological processes including cell-matrix interactions, osteoblast differentiation, embryonic development, and homeostasis. SMOC-1 was found to be elevated in asymptomatic Alzheimer’s disease (AD) patient cortex as well as being enriched in amyloid plaques and in AD patientcerebrospinal fluid, arguing for SMOC-1 as a promising biomarker for AD. Having access to high-quality SMOC-1 antibodies is crucial for the scientific community. It can ensure the consistency and reliability of SMOC-1 research, and further the exploration of its potential as both a therapeutic target or diagnostic marker.. In this study, we characterized seven SMOC-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. We identified successful antibodies in the tested applications and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.


Introduction
2][3] Expressed in various tissues with localization to the basement membrane and extracellular matrix, SMOC-1 regulates cell-matrix interactions through its ability to bind cell-surface receptors, growth factors, extracellular matrix and cytokines. 2,4Through its binding to receptors on the cells surface, SMOC-1 modulates growth factor signalling involved in osteoblast differentiation. 5In addition to being a critical regulator of various biological processes, SMOC-1 plays a role in the pathophysiology of diverse diseases, including cancer development and progression. 1oteomic studies have uncovered SMOC-1 to be highly enriched in a subpopulation of amyloid plaques, in AD patients and to be elevated in asymptomatic AD cortex. 6Recently, SMOC-1 was shown to be elevated in cerebrospinal fluid from AD patients. 7Although it remains unknown why SMOC-1 co-localizes with only some amyloid plaques, it is hypothesized that SMOC-1 may interact with amyloid-beta (Aβ) species that have been subjected to post-translational modifications. 6More comprehensive research is required to examine the mechanistic role of SMOC-1 in AD.
Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.
Here we evaluated the performance of seven commercial antibodies for SMOC-1 for use in western blot and immunoprecipitation, enabling biochemical and cellular assessment of SMOC-1 properties and function.The platform for antibody characterization used to carry out this study was endorsed by a committee of industry academic representatives.It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterization procedures using most commercially available antibodies against the corresponding protein.The standardized consensus antibody characterization protocols are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1). 8e authors do not engage in result analysis or offer explicit antibody recommendations.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.Guidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway. 9

Results and discussion
1][12][13][14] To identify a cell line that expresses adequate levels of SMOC-1 protein to provide sufficient signal to noise, we examined public proteomics databases, namely PaxDB 15 and DepMap. 16HeLa was identified as a suitable cell line and thus HeLa was modified with CRISPR/Cas9 to knockout the corresponding SMOC1 gene (Table 1).SMOC-1 is predicted to be a secreted protein.Accordingly, we collected concentrated culture media from both parental and SMOC1 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 the tested antibodies are shown in Figures 1 and 2.

REVISED Amendments from Version 1
To this revised version of the article, the authors have provided an additional description of the YCharOS antibody characterization platform as well as references to the published standardized protocols and feature article that guides readers on how to interpret the characterization data.This background information has been included to the Introduction.
Additionally, the authors have indicated, in the figure legends, which secondary antibodies or detection system was used.
Any further responses from the reviewers can be found at the end of the article  1 refers to new antibodies with RRID that have recently been created (August 2023) but will be available on the Antibody Registry in the coming weeks.
In conclusion, we have screened seven SMOC-1 commercial antibodies by Western blot and immunoprecipitation.Under our standardized experimental conditions, several high-quality antibodies were identified, however, the authors do not engage in result analysis or offer explicit antibody recommendations.A limitation of this study is the use of universal protocols -any 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.
The underlying data can be found on Zenodo, an open-access repository. 17,18thods Antibodies All SMOC-1, antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (RRID), to ensure the antibodies are cited properly. 19Peroxidase-conjugated goat anti-rabbit is from Thermo Fisher Scientific (cat.number 65-6120).CRISPR/Cas9 genome editing HeLa SMOC1 KO clone was generated with low passage cells using an open-access protocol available on Zenodo.org.The guide RNA used to knockout the SMOC1 gene is CUCGUAGGACCUGCCAUCAG.

Cell culture
Both HeLa WT and SMOC1 KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly. 20 Antibody screening by Western blot on culture media HeLa cells WT and SMOC1 KO were washed three times with PBS 1x and starved for ~18 hrs.Culture media were collected and centrifuged for 10 min at 500 x g to eliminate cells and larger contaminants, then for 10 min at 4500 x g to eliminate smaller contaminants.Culture media were concentrated by centrifuging at 4000 x g for 30 min using Amicon Ultra-15 Centrifugal Filter Units with a membrane NMWL of 10 kDa (MilliporeSigma cat.number UFC901024).Culture media were supplemented with 1x protease inhibitor cocktail mix (MilliporeSigma, cat.number P8340).
14]21 Western blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific (cat.number WXP42012BOX) ran with Tris/Glycine/SDS buffer from Bio-Rad (cat.number 1610772), loaded in Laemmli loading sample buffer from Thermo Fisher Scientific (cat.number AAJ61337AD) and transferred on nitrocellulose membranes.BLUelf prestained protein ladder from GeneDireX (cat.number PM008-0500) was used.Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat.number BP103-10) 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% milk in TBS with 0,1% Tween 20 (TBST) (Cell Signalling Technology, cat.number 9997).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 were incubated with Pierce ECL from Thermo Fisher Scientific (cat.number 32106) prior to detection with the iBright™ CL1500 Imaging System from Thermo Fisher Scientific (cat.number A44240).
14]22 Antibody-bead conjugates were prepared by adding 2 μg of antibody to 500 μL of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat.number 87788) in a 1.5 mL microcentrifuge tube, together with 30 μL of Dynabeads protein A-(for rabbit antibodies) from Thermo Fisher Scientific (cat.number 10002D).Tubes were rocked for ~1 hr at 4°C followed by two washes to remove unbound antibodies.
Starved HeLa WT culture media were concentrated as described above and supplemented with protease inhibitor.0.3 mL aliquots at 1.6 mg/mL of protein were incubated with an antibody-bead conjugate for ~1 hr at 4°C.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 a precast midi 4-20% Tris-Glycine polyacrylamide gels.VeriBlot for IP Detection Reagent: HRP from Abcam (cat.number ab131366) was used as a secondary detection system at a concentration of 0. the authors do not provide a detailed analysis and interpretation of their results, the data are well presented and clearly indicate suitable antibodies for WB and IP studies.Additional comments: 1) It would have been helpful if the authors had explained why immunofluorescence or immunohistochemistry were not considered for this study.
2) It would be useful to also indicate in the figure captions that a secondary antibody was used.
3) It might be understandable that the authors do not make explicit antibody recommendations.However, the controversial result of the secondary reagent used for IP should be discussed.VeriBlot for IP detection reagent was most likely used to avoid interference from denatured IgG.Nevertheless, the heavy chain was detected in some antibodies.
4) In the methods section on immunoprecipitation, the authors should clarify how samples were processed for SDS-PAGE and Western blotting.

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

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: antibody alternative scaffolds, phage display 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.
immunohistochemistry were not considered for this study.Following our standardized antibody characterization platform, available on Protocol exchange (https://doi.org/10.21203/rs.3.pex-2607/v1),immunofluorescence (IF) is not performed when the target protein is secreted.Given the fixation and permeabilization methods used in this protocol, performing IF for secreted proteins presents many limitations and challenges.Immunohistochemistry (IHC) is not currently within the characterization platform.The main reason being that, given our knockout (KO) cell line-based protocol, it would be extremely challenging to KO proteins in human tissues for the entire human genome.That being said, the idea of including IHC to the platform is being troubleshooted and we hope to include this antibody-based application in the future.We've recently included Flow Cytometry (FC) to our protocol and are in the process of revisiting many Data Notes to include the FC data.

2) It would be useful to also indicate in the figure captions that a secondary antibody was used.
Thank you for this feedback.
We will be submitting a new version of this manuscript and the secondary antibodies and detection systems used will be included in the figure legends for all applications.
3) It might be understandable that the authors do not make explicit antibody recommendations.However, the controversial result of the secondary reagent used for IP should be discussed.VeriBlot for IP detection reagent was most likely used to avoid interference from denatured IgG.Nevertheless, the heavy chain was detected in some antibodies.
VeriBlot was used as a secondary detection system as the expected SMOC-1 molecular weight was very similar to that of the immunoglobulin heavy chain, 48 kDa and 50 kDa, respectively.The proximity of these molecular weights can cause antibodies used in the WB following the IP to cross-react with the heavy chain, preventing the true immunoprecipitated signal from being detected.That is why VeriBlot was used as the secondary detection systems as it would not react with the heavy chain.This is proven correct as the heavy chain is detected independently in the IP of 3 antibodies (ab200219, PA5-113408 and A20482).
To understand our selection method of secondary detection systems for IP experiments, please refre to Figure 9 of the detailed procedure on Protocol exchange ( https://doi.org/10.21203/rs.3.pex-2607/v1), that will be listed as reference 8 in the updated version of the article.

4) In the methods section on immunoprecipitation, the authors should clarify how samples were processed for SDS-PAGE and Western blotting.
In the updated version of this manuscript, to be submitted shortly, we will include the reference to our entire antibody characterization platform which provides the step-by-step details of this procedure.
Competing Interests: No competing interests were disclosed.

Deborah Moshinsky
Institute for Protein Innovation, Boston, Massachusetts, USA SPARC (secreted protein acidic and rich in cysteine)-related calcium-binding protein 1 (SMOC-1) is involved in the pathophysiology of a number of diverse diseases.In Alzheimer's disease (AD), SMOC-1 has been found to co-localize with some amyloid plaques, although it's mechanistic role in the disease has not been well-investigated.The availability of high quality antibodies would facilitate research into SMOC-1's involvement in AD, so the investigators compared the performance of 7 commercially available antibodies in Western blot and immunoprecipitation to allow researchers to assess antibody performance in these applications.
The authors present a scientifically sound Western blot and immunoprecipitation study of all 7 antibodies against SMOC-1.The protocols were presented in sufficient detail for the work to be repeated and the reader can interpret whether the antibodies worked or not under the given experimental conditions.No assessment of the results was given, however the authors give appropriate reasoning for not including such an assessment.

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: antibody characterization and validation
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.The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com blot; IF= immunofluorescence; IP=immunoprecipitation. **= recombinant antibody.

Figure 2 .
Figure2.SMOC-1 antibody screening by immunoprecipitation on culture media.Immunoprecipitation was performed on concentrate culture media from HeLa WT, and using 2.0 μg of the indicated SMOC-1, antibodies precoupled to Dynabeads protein A. Samples were washed and processed for Western Blot with the indicated SMOC-1, antibody.For Western blot, ab313569** was used at 1/1000.The Ponceau stained transfers of each blot are shown for similar reasons as in Figure1.VeriBlot for IP Detection Reagent:HRP was used as a secondary detection system.SM=8% starting material; UB=8% unbound fraction; IP=immunoprecipitated, HC= antibody heavy chain, **= recombinant antibody.

Figure 1 .
Figure 1.SMOC-1 antibody screening by Western blot on culture media.HeLa WT and SMOC1 KO were cultured in serum free media, and 30 μg of protein from concentrated culture media were processed for Western blot with the indicated SMOC-1, antibodies.The Ponceau stained transfers of each blot are shown.Peroxidase-conjugated goat anti-rabbit was used as the secondary antibody to detect the signal produced.Antibody dilutions were chosen according to the recommendations of the antibody supplier.All antibodies were tested at 1/2000.Predicted band size: 48 kDa.**= recombinant antibody.

Table 1 .
Summary of the cell lines used.
3 μg/mL.NeuroSGC/YCharOS/EDDU collaborative group: Riham Ayoubi, Thomas M. Durcan, Aled M. Edwards, Carl Laflamme, Peter S. McPherson, Chetan Raina and Kathleen Southern Thank you to the Structural Genomics Consortium, a registered charity (no.1097737), for your support on this project.The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no.OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no.875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.An earlier version of this article can be found on Zenodo (doi: 10.5281/zenodo.8277962).