A guide to selecting high-performing antibodies for TMEM175 (UniProt ID: Q9BSA9) for use in western blot, immunoprecipitation, and immunofluorescence

Introduction

TMEM175 is the pore-forming subunit of a lysosomal K+ channel complex with AKT, located on endosomes and lysosomes.^1,2 Under certain conditions, it exhibits H⁺ permeability,³ regulating proton efflux to maintain lysosomal homeostasis.⁴ Additionally, knocking out TMEM175 disrupts the fusion of lysosomes with autophagosomes.¹ Mutations in the TMEM175 gene present high-risk factors for neurodegenerative diseases such as Parkinson’s.⁵

This research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data.⁶ 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.⁶ Here we characterized six commercial TMEM175 antibodies, selected and donated by participant antibody manufacturers, for use in western blot, immunoprecipitation, and immunofluorescence, enabling biochemical and cellular assessment of TMEM175 properties and function.

The 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 gateway⁷ and in Table 3 of this data note.⁶

Results and discussion

Our standard protocol involves comparing readouts from wild type (WT) and KO cells.^8,9 The first step was to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal using antibodies. To this end, we examined the DepMap (Cancer Dependency Map Portal, RRID:SCR_017655) 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.¹⁰ HeLa expresses the TMEM175 transcript at 3.8 log₂ TPM + 1. A TMEM175 KO HeLa cell line was obtained from Abcam ( Table 1).

To screen all six by western blot, WT and TMEM175 KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with the TMEM175 antibodies in parallel ( Figure 1A). TMEM175 protein expression was also investigated by western blot ( Figure 1B) using lysates from cell lines ( Table 1) with various RNA levels indicated in Figure 1B.

Figure 1. TMEM175 antibody screening by western blot.

A) Lysates of HeLa WT and TMEM175 KO were prepared, and 20 μg of protein were processed for western blot with the indicated TMEM175 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. Antibody dilutions used: ab300457** at 1/1000, ab309571** at 1/200, ab309572** at 1/1000, 19925–1-AP at 1/1000, 68172–1-Ig* at 1/5000, and 703805** at 1/1000. Antibodies ab309571** and ab309572** were not recommended for western blotting and were initially tested at 1/1000 then titrated to improve signal when needed. B) Lysates of HeLa WT and TMEM175 KO as well as A172, DM S53, U-87 MG and SH-SY5Y were prepared, and 20 μg of protein were processed for western blot with anti-TMEM175 ab309572** diluted at 1/1000. The unit of the indicated RNA values is log₂ TPM + 1. Predicted band size: 55.6 kDa. **Recombinant antibody; *Monoclonal antibody.

We then assessed the capability of the six antibodies to capture TMEM175 from HeLa protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific TMEM175 antibody identified previously (refer to Figure 1A) was selected. Equal amounts of the starting material (SM) and the unbound fractions (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE ( Figure 2).

Figure 2. TMEM175 antibody screening by immunoprecipitation.

HeLa lysates were prepared, and immunoprecipitation was performed using 1 mg of lysate and 2.0 μg of the indicated TMEM175 antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the TMEM175 antibody ab309572** diluted at 1/1000. The Ponceau stained transfers of each blot are shown. SM = 4% starting material; UB = 4% unbound fraction; IP = immunoprecipitate, HC = antibody heavy chain. **Recombinant antibody; *Monoclonal antibody.

For immunofluorescence, the six antibodies were screened using a mosaic strategy. First, HeLa WT and TMEM175 KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the TMEM175 antibodies were evaluated. Both WT and KO lines imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 3). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested, and the images presented in Figure 3 are representative of this analysis.⁶

Figure 3. TMEM175 antibody screening by immunofluorescence.

HeLa WT and TMEM175 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 indicatedTMEM175 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 green and magenta dashed line, respectively. When an antibody was recommended for immunofluorescence by the supplier, we tested it at the recommended dilution. The rest of the antibodies were tested at 1 and 2 μg/ml, and the final concentration was selected based on the detection range of the microscope used and a quantitative analysis not shown here. Antibody dilutions used: ab300457** at 1/250, ab309571** at 1/500, ab309572** at 1/500, 19925–1-AP at 1/300, 68172–1-Ig* at 1/500, and 703805** at 1/100. Bars = 10 μm. **Recombinant antibody; *Monoclonal antibody.

In conclusion, we have screened six TMEM175 commercial antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human HeLa WT and TMEM175 KO cells. To assist users in interpreting antibody performanyce, Table 3 outlines various scenarios in which antibodies may perform in all three applications.¹⁰ Several high-quality and renewable antibodies that successfully detect TMEM175 were identified in all applications. Researchers who wish to study TMEM175 in a different species are encouraged to select high-quality antibodies, based on the results of this study, and investigate the predicted species reactivity of the manufacturer before extending their research.

Limitations

Inherent limitations are associated with the antibody characterization platform used in this study. Firstly, the YCharOS project focuses on renewable (recombinant and monoclonal) antibodies and does not test all commercially available TMEM175 antibodies. YCharOS partners provide approximately 80% of all renewable antibodies, but some top-cited polyclonal antibodies may not be available through these partners.

Secondly, the YCharOS effort employs a non-biased approach that is agnostic to the protein for which antibodies have been characterized. The aim is to provide objective data on antibody performance without preconceived notions about how antibodies should perform or the molecular weight that should be observed in western blot. As the authors are not experts in TMEM175, only a brief overview of the protein’s function and its relevance in disease is provided. TMEM175 experts are invited to analyze and interpret observed banding patterns in western blots and subcellular localization in immunofluorescence.

Thirdly, YCharOS experiments are not performed in replicates primarily due to the use of multiple antibodies targeting various epitopes. Once a specific antibody is identified, it validates the protein expression of the intended target in the selected cell line, confirms the lack of protein expression in the KO cell line and supports conclusions regarding the specificity of the other antibodies. All experiments are performed using master mixes, and meticulous attention is paid to sample preparation and experimental execution. In IF, the use of two different concentrations serves to evaluate antibody specificity and can aid in assessing assay reliability. In instances where antibodies yield no signal, a repeat experiment is conducted following titration. Additionally, our independent data is performed subsequently to the antibody manufacturers internal validation process, therefore making our characterization process a repeat.

Lastly, as comprehensive and standardized procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a single cell type for evaluating antibody performance poses as a limitation, as factors such as target protein abundance significantly impact results. Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations can impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines.

Method

The standardized protocols used to carry out this KO cell line-based antibody characterization platform was established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterize antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on Protocols.io (protocols.io/view/a-consensus-platform-for-antibody-characterization ).⁶ Brief descriptions of the experimental setup used to carry out this study can be found below.

Cell lines and antibodies

Cell lines used and primary antibodies tested in this study are listed in Tables 1 and 2, respectively. To ensure that the cell lines and antibodies are cited properly and can be easily identified, we have included their corresponding Research Resource Identifiers, or RRID.^11,12

Table 2. Summary of the TMEM175 antibodies tested.

Company	Catalog number	Lot number	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μL)	Vendors recommended applications
Abcam	ab300457**	1073064–1	AB_3101786	recombinant mono	EPR24415–47	rabbit	0.49	Wb
Abcam	ab309571**	1057532-2	AB_3105951	recombinant mono	EPR26739–94	rabbit	1.03	other
Abcam	ab309572**	1057536-2	AB_3105952	recombinant mono	EPR24415–47	rabbit	1.05	other
Proteintech	19925–1-AP	00138879	AB_10666166	polyclonal	-	rabbit	0.60	Wb, IP, IF
Proteintech	68172–1-Ig*	10028030	AB_2923687	monoclonal	1E7E1	mouse	1.00	Wb
Thermo Fisher Scientific	703805**	2874381	AB_2866474	recombinant mono	5H18L65	rabbit	0.50	Wb, IF

** Recombinant antibody.

* Monoclonal antibody.

Table 3. Illustrations to assess antibody performance in all western blot, immunoprecipitation and immunofluorescence.

Western blot	Immunoprecipitation	Immunofluorescence

Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are (Thermo Fisher Scientific, cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies (Thermo Fisher Scientific, cat. number A-21429 and A-21424). Peroxidase-conjugated Protein A for IP detection is from Cell Signaling Technology, cat. number 12291.