The identification of high-performing antibodies for TDP-43 for use in Western Blot, immunoprecipitation and immunofluorescence

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA binding protein playing a critical role in the regulation of transcription, splicing and RNA stability. Mutations in TARDBP leading to aggregation, are suspected to be a characteristic feature of various neurogenerative diseases. The lack of well-characterized anti- TDP-43 antibodies acts as a barrier to establish reproducible TDP-43 research. In this study, we characterized eighteen TDP-43 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 well-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Introduction TDP-43, encoded by the TARDBP gene, is a DNA/RNA-binding protein implicated in RNA metabolism and processing. 1 Belonging to the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins that bind to RNA via highly conserved RNA recognition motifs, TDP-43 binds to UG-repeats with high specificity. 1,2 Mutations in TARDBP that result in TDP-43 aggregation and neuropathology have been observed in distinct neurodegenerative diseases, known as TDP-43 proteinopathies. 3,4 Various studies have identified a subset of amyotrophic lateral sclerosis (ALS) patients that possess TARDBP mutations, suggesting that TDP-43 gain of toxic function or loss of function is a causative factor in sporadic and/or familial ALS. [4][5][6] Mechanistic studies would be greatly facilitated by the availability of high-quality antibodies.
Here, we compared the performance of a range of commercially available antibodies for TDP-43 and characterized highquality antibodies for Western blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of TDP-43 properties and function.

Results and discussion
Our standard protocol involved comparing readouts from wild-type (WT) and TARDBP knockout (KO) cells. 7,8 The first step was to identify a cell line(s) that expresses sufficient levels of TDP-43 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 HAP1 cells expressed the TARDBP transcript at RNA levels above the average range of cancer cells analyzed. Parental and TARDBP knockout HAP1 cells were obtained from Horizon Discovery (Table 1).
For Western Blot experiments, we resolved proteins from WT and TARDBP KO cell extracts and probed them side-byside with all antibodies in parallel ( Figure 1).
For immunoprecipitation experiments, we used the antibodies to immunopurify TDP-43 from HAP1 cell extracts. The performance of each antibody was evaluated by detecting the TDP-43 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates ( Figure 2). 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 staining, imaging and image analysis bias ( Figure 3).
In conclusion, we have screened TDP-43 commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence and identified several high-performing antibodies under our standardized experimental conditions.

Antibodies
All TDP-43 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (RRID), to ensure the antibodies are cited properly. 10 Peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies are from

REVISED Amendments from Version 1
In this new version of the article, we have added the DOI to references 9,12 and 13. This is to make it easier for readers to access the citations.

Cell culture
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 Signaling 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 ECL (Thermo Fisher Scientific, cat. number 32106) prior to detection with the iBright™ CL1500 Imaging System (Thermo Fisher Scientific, cat. number A44240).
Antibody screening by immunoprecipitation Immunoprecipitation was performed as described in our standard operating procedure. 13 Antibody-bead conjugates were prepared by adding 2 μg 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) or protein G-(for mouse antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Tubes were rocked for~2 hrs at 4°C followed by two washes to remove unbound antibodies.
HAP1 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) (Thermo Fisher Scientific, cat. number 87788), supplemented with protease inhibitor (Millipore Sigma, cat. number P8340). Lysates were rocked for 30 min at 4°C and spun at 110,000Â g for 15 min at 4°C. 0.5 ml aliquots at 2.0 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 precast midi 4-20% Tris-Glycine polyacrylamide gels. Prot-A: HRP (MilliporeSigma, cat. number P8651) was used as a secondary detection system at a dilution of 0.4 μg/ml for an experiment where a rabbit antibody was used for both immunoprecipitation and its corresponding Western blot.
Pathological TDP-43 is generally aggregated, fragmented, and hyperphosphorylated, which may affect antibody recognition. Thus, although it would be beyond the scope of this study, future validation using human patients' samples, as recommended by the reviewer #1, or of the recognition sites of each antibody would make this data even more valuable.
The references 9, 12, and 13 were found to be available on Zendo, but it is difficult to reach them because of a lack of direct links. It is recommended that the DOI be appended.

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?