Identification of high-performing antibodies for tyrosine-protein kinase SYK for use in Western Blot, immunoprecipitation and immunofluorescence

Tyrosine-protein kinase SYK, encoded by the SYK gene, is a non-receptor type protein kinase which mediates immune signal transduction through immunoreceptors. Tyrosine-protein kinase SYK expression has been associated with the development of various inflammatory diseases, cancer and neurodegenerative conditions. The reproducibility of tyrosine-protein kinase SYK research would help elucidate the mechanism in which it causes neuroinflammation as well as its potential as a novel target to treat Alzheimer’s disease. This would be facilitated with the availability of high-quality tyrosine-protein kinase SYK. In this study, we characterized thirteen tyrosine-protein kinase SYK 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.


Introduction
Tyrosine-protein kinase SYK, also known as spleen tyrosine kinase (SYK), is a non-receptor type of protein-tyrosine kinase (PTK) predominantly recognized for its role in amplifying immune responses. 1Unique to other families of PTKs, SYK has tandem N-terminal Src homology 2 (SH2) domains as well as a tyrosine kinase domain at its C-terminal region.[3] Functioning as a vital mediator of cellular inflammatory responses, SYK is primarily known to contribute to allergies, 4 autoimmune diseases 5,6 and B-cell malignancies. 7Emerging research has suggested SYK may be implicated in the development in neuroinflammatory symptoms that are characteristic of Alzheimer's disease (AD). 8,9As such, studies have demonstrated that inhibition or down-regulation of SYK increases amyloid-beta (Aβ) clearance and decreases Tau hyperphosphorylation, highlighting it's potential as a therapeutic target to treat AD. [9][10][11] Further investigation is required to elucidate the mechanism in which SYK influences Tau pathology and Aβ deposition. 8chanistic 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 tyrosine-protein kinase SYK and validated several antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of tyrosine-protein kinase SYK properties and function.
For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy. 25In brief, we plated WT and SYK KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis bias (Figure 3).
In conclusion, we have screened tyrosine-protein kinase SYK commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies under our standardized experimental conditions.Under our standardized experimental conditions, several high-quality antibodies were identified, however,

REVISED Amendments from Version 2
In the latest version of this article, we have clarified the selection process antibodies were selected for Western Blots assessment of antibody performance in the immunoprecipitation experiments.Additionally, we have included the method used to determine protein concentration of the lysates.
Any further responses from the reviewers can be found at the end of the article 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 supporting this study is found on Zenodo, an open access repository. 26,27

Antibodies
All tyrosine-protein kinase SYK antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly. 28Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat.number 65-6120 and 62-6520).Alexa-555-conjugated goat antirabbit and anti-mouse secondary antibodies are from Thermo Fisher Scientific (cat.number A21429 and A21424).The AFFN-SYK-5A10* antibody was deposited to the Developmental Studies Hybridoma Bank (DSHB) by EU Program Affinomics (DSHB Hybridoma Product AFFN-SYK-5A10).

Cell culture
Both THP-1 WT and SYK KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly. 29

Antibody screening by Western Blot
Western Blots were performed as described in our standard operating procedure. 30Protein concentration of the lysates was measured using a BCA protein assay kit.The resulting protein concentrations was adjusted to ensure equal amounts of protein in the WT and KO lysates were loaded in each lane.
THP-1 WT and SYK KO were collected in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific, cat.number 89901) supplemented with protease inhibitor.Lysates were sonicated briefly and incubated for 30 min on ice.Lysates were spun at ~110,000 Â g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western Blot.BLUelf prestained protein ladder (GeneDireX, cat.number PM008-0500) was used.Western Blots were performed with large 5-16% gradient polyacrylamide gels and transferred on nitrocellulose membranes.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 Blots.Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin (BSA) (Wisent, cat.number 800-095) 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 (Thermo Fisher Scientific, cat.number 32106) prior to detection with the HyBlot CL autoradiography films (Denville, cat.number 1159T41).

Antibody screening by immunoprecipitation
Immunoprecipitation was performed as described in our standard operating procedure. 31Antibody-bead conjugates were prepared by adding 2 μg or 10 μL of antibody at an unknown concentration to 500 μL of Pierce IP Lysis Buffer (Thermo Fisher Scientific, cat.number 87788) in a 1.5 mL microcentrifuge tube, together with 30 μL of Dynabeads protein A -(for rabbit antibodies) or protein G -(for mouse antibodies) (Thermo Fisher Scientific, cat.number 10002D and 10004D).Tubes were rocked for ~2 hrs at 4°C followed by several washes to remove unbound antibodies.
THP-1 WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) supplemented with protease inhibitor.Lysates were rocked 30 min at 4°C and spun at 110,000 Â g for 15 min at 4°C.One mL aliquots at 1.0 mg/mL of lysate were incubated with an antibody-bead conjugate for ~2 hrs 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 5-16% gradient polyacrylamide gels.Refers to RRID that were recently created (July 2023) and will be added to the Antibody Registry in the coming weeks.
Imaging was performed using a Zeiss LSM 700 laser scanning confocal microscope equipped with a Plan-Apo 20x air objective (NA = 0.8
The data note entitled "Identification of high-performing antibodies for tyrosine-protein kinase SYK for use in Western Blot, immunoprecipitation, and immunofluorescence" compares thirteen commercial antibodies against tyrosine-protein kinase SYK using three ○ different immunodetection techniques to provide relevant data regarding the sensitivity and specificity of these antibodies for SYK protein tyrosine kinase.My main criticism is that the results and discussion section is little developed; for example, are not briefly described the most relevant results of any of the three experiments and are not discussed the results that were different, for example, why the GTX633910 antibody detects a polypeptide band above 75 kDa while the 72 kDa band corresponding to the Tyrosine-protein kinase SYK is very poorly detected; why is not detected with the AFFN-SYK-5A10 antibody the Tyrosine-protein kinase SYK; why in immunoprecipitation the and ab244968 antibodies were used to immunodetect the unbound fraction and not other antibodies.The authors should provide answers in the text to these questions.

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In immunofluorescence results, place the white light micrograph of the cells next to the fluorescence image to observe the cell outline is recommended and can then be followed by the colored segmented lines in the fluorescence image.

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Write in the protocol how the total protein concentration in the supernatant was determined and how many the volume in microliters used to obtain 30 mg of protein from WT cells as from SYK KO cells in each lane to do better reproducible the experiment.
○ Also the conclusion is observed underdeveloped.I recommend the authors to conclude by offering their own recommendations on bioanalytical applications of the antibodies analyzed and compare with the vendor-recommended applications read in Table 2.

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: Structure and function of protein kinases A
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.
kinase SYK for use in Western Blot, immunoprecipitation, and immunofluorescence" compares thirteen commercial antibodies against tyrosine-protein kinase SYK using three different immunodetection techniques to provide relevant data regarding the sensitivity and specificity of these antibodies for SYK protein tyrosine kinase.My main criticism is that the results and discussion section is little developed; for example, are not briefly described the most relevant results of any of the three experiments and are not discussed the results that were different, for example, why the GTX633910 antibody detects a polypeptide band above 75 kDa while the 72 kDa band corresponding to the Tyrosine-protein kinase SYK is very poorly detected; why is not detected with the AFFN-SYK-5A10 antibody the Tyrosine-protein kinase SYK; why in immunoprecipitation the 80460 and ab244968 antibodies were used to immunodetect the unbound fraction and not other antibodies.The authors should provide answers in the text to these questions.

○
While the authors understand your concern regarding the lack of result analysis, we must re-iterate, that the authors do not engage in result analysis nor offer explicit antibody recommendations.The format of this article is a Data Note, with the goal being to provide brief descriptions of research data for potential reuse or to benefit for the scientific community without providing any analyses or conclusions, as explained in their article guidelines (https://f1000research.com/for-authors/article-guidelines/data-notes).The GTX633910 antibody detecting bands above the 75 kDa molecular marker in both the wild-type (WT) and the knockout (KO) could indicate that this antibody is non-selective, binding to epitopes other than the intended target.The Western blot assessment of antibodies in the unbound fraction as well as the immunoprecipitation are carried out with antibodies previously KO-validated by Western blot (Figure 1), which is why antibodies 8060 and ab244968 were selected.We understand that this last point was not made clear in the text.In the newly submitted version of this article we have included a statement in the results and discussion section to clarify this part of the protocol.
In immunofluorescence results, place the white light micrograph of the cells next to the fluorescence image to observe the cell outline is recommended and can then be followed by the colored segmented lines in the fluorescence image.

○
With our immunofluorescence protocol described in the methods section of this article, the goal is not to define the cellular location of the target protein but rather to determine where or not ratio of signal between the WT and KO cells is significantly different.This is why we outline of the cell is not as significant compared to the signal produced by the cells themselves.
Write in the protocol how the total protein concentration in the supernatant was determined and how many the volume in microliters used to obtain 30 mg of protein from WT cells as from SYK KO cells in each lane to do better reproducible the experiment.

○
The protocol describing how the total protein concentration was determined as well as the method to obtain 30 mg of protein has been included in the Methods section describing Antibody screening by Western blot.Also the conclusion is observed underdeveloped.I recommend the authors to conclude by offering their own recommendations on bioanalytical applications of the antibodies analyzed and compare with the vendor-recommended applications read in Table 2.

○
The authors to not offer their own recommendations nor compare the antibody performance in each application to the vendors recommended application.The reason being that the antibodies are tested under one specific set of conditions, therefore summarizing the performance of the antibodies would only be valid under these precise experimental set-up and cell lines used.The goal of the YCharOS initiative is to present antibody characterization data to the scientific community, using a standardized protocol that allows researchers to select high-performing antibodies.This enables researchers who specialize in the target of interest, in this case Tyrosine -protein kinase SYK, to conduct further studies.This intention is clearly defined in the concluding paragraph of the results and discussion section.
We hope that with our clarifications, responses to your concerns, and the statements included in the latest version of the article, you no longer have reservations about the status of this article.The presented antibody characterization data will be highly beneficial for researchers aiming to enhance their studies on tyrosine-protein kinase SYK.
Competing Interests: No competing interests were disclosed.

Kiyonao Sada
University of Fukui, Fukui, Japan The importance of the protein-tyrosine kinase Syk has been highlighted in new research areas for studying the pathogenesis and developing treatments of neurological diseases.In this study, the authors compare the usefulness of anti-Syk antibody-based approaches using wild-type and Sykdeficient THP-1 cells, established by genome editing.
It is very important to know which antibodies commercially available are useful for a particular protein, not just Syk.This paper does not indicate which antibodies are recommended, but allows experts reading this paper to make their own interpretations.The experimental conditions are based on the recommended dilution factors, and in practice, further investigation by the researcher is required.However, it is not practical for many researchers to purchase and compare all antibodies, so the Syk results provide very important information.
Western blot results were relatively uniform, but differences in the usefulness of each antibody were observed in immunoprecipitation and immunofluorescence.In particular, the reader is provided with the information that some antibodies could detect Syk effectively despite the lack of company recommendations for immunofluorescence.
Regarding Table 2, in discussing the experimental results, it is also necessary to present information about the antigens of these antibodies.This is because it is assumed that when immunoprecipitation is performed, the antibodies mask the antigenic sites, making it difficult to detect proteins that associate with the same sites.
Overall, I can conclude that this is a very useful paper for researchers who are pioneering new studies on Syk.
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?Partly Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Signal transduction 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.
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Figure 2 .
Figure 2. Tyrosine-protein kinase SYK antibody screening by immunoprecipitation.THP-1 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated tyrosine-protein kinase SYK antibodies pre-coupled to Dynabeads protein G or protein A. A) Ability of the antibodies to immunocapture tyrosine-protein kinase SYK was first assessed by comparing the level of protein available in the starting material to the level remaining in the unbound fractions.B) The immunoprecipitates for antibodies which would immunocapture tyrosine-protein kinase SYK in (A) are shown.For Western Blot, 80460* and ab244968** were used at 1/3000.The Ponceau stained transfers of each blot are shown.SM = 10% starting material; UB = 10% unbound fraction; IP = immunoprecipitated.*Monoclonal antibody, **Recombinant antibody.

Reviewer
Report 03 January 2024 https://doi.org/10.5256/f1000research.157120.r228375© 2024 Sada K.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Table
. Summary of the cell lines used.
).All cell images represent a single focal plane.Figures were assembled with Adobe Photoshop (version 24.1.2) to adjust contrast then assembled with Adobe Illustrator (version 27.3.1).