The identification of high-performing antibodies for transmembrane protein 106B (TMEM106B) for use in Western blot, immunoprecipitation, and immunofluorescence

Transmembrane protein 106B (TMEM106B), a protein that is localized to the lysosome, is genetically linked to many neurodegenerative diseases and forms fibrils in diseased brains. The reproducibility of TMEM106B research would be enhanced if the community had access to well-characterized anti-TMEM106B antibodies. In this study, we characterized six commercially available TMEM106B antibodies for their performance in 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
Transmembrane protein 106B (TMEM106B) is a genetic risk variant for many neurodegenerative diseases. The presence of the TMEM106B major risk allele, rs1990622, is suspected to be a risk factor and disease modifier for Frontotemporal Dementia (FTD), with few studies investigating its potential role in Amyotrophic Lateral Sclerosis (ALS) pathogenesis. [1][2][3][4][5] TMEM106B is a transmembrane endosomal and lysosomal glycoprotein. The protein has garnered interest lately, with the discovery that a 135 amino acid portion of the protein from its luminal C-terminal domain forms fibrils in the brains of patients with frontotemporal lobar degeneration, progressive supranuclear palsy, and dementia with Lewy bodies. 6,7 The roles of TMEM106B fibrils in normal lysosomal function or disease pathogenesis are not known, nor is the mechanism by which the protein is proteolyzed, or forms fibrils. 7 Mechanistic studies would be greatly facilitated with the availability of high-quality validated antibodies. Here, we compared the performance of a range of commercially available antibodies for TMEM106B and characterized several high-quality antibodies for Western blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of TMEM106B properties and function.

Results and discussion
Our standard protocol involved comparing readouts from wild-type (WT) and knockout (KO) cells. 8,9 The first step was to identify a cell line(s) that expresses sufficient levels of TMEM106B to generate a measurable signal. To this end, we examined the DepMap transcriptomics databases 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 TMEM106B at RNA levels above the average range of cancer cells analyzed. The parental and KO HAP1 cell lines were obtained from Horizon Discovery (Table 1).
For Western blot experiments, we resolved proteins from WT and TMEM106B KO cell extracts and probed them side-byside with all antibodies in parallel ( Figure 1).   . Transmembrane protein 106B antibody screening by immunofluorescence. HAP1 WT and TMEM106B 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 in a 96-well plate with a glass bottom. Cells were stained with the indicated TMEM106B antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels were 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 the concentration was not indicated by the supplier, we tested antibodies at 1/1000 or 1/2000. At this concentration, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab244516 at 1/100, A20165 at 1/2000, 93334** at 1/100, 60333-1-lg* at 1/2000, PA5-34353 at 1/1000, and PA5-63558 at 1/100. Bars = 10 μm. *= monoclonal antibody, **= recombinant antibody.

60333-1-lg
For immunoprecipitation experiments, we used the antibodies to immunopurify TMEM106B from HAP1 cell extracts. The performance of each antibody was evaluated by detecting the TMEM106B 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. 10 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 many TMEM106B commercial antibodies by Western blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies under our standardized experimental conditions.

Antibodies
All TMEM106B antibodies are listed in Table 2 together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly. 11 Peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies are from Thermo Fisher Scientific (cat. number 62-6520 and 65-6120). Alexa-555 conjugated secondary goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424) Cell culture Both HAP1 WT and TMEM106B KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly. 12

Antibody screening by Western blot
Western blot experiments were performed as described in our standard operating procedure. 13 HAP1 WT and TMEM106B KO were collected in RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) from Thermo Fisher Scientific (cat number 0089901), supplemented with 1x protease inhibitor cocktail mix from MilliporeSigma (cat. number 78429). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at~110,000 x 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 from GeneDireX (cat. number PM008-0500) was used.
Western blots were performed with pre-cast mini 4-15% gradient polyacrylamide gels from Bio-Rad (cat. number 4561084) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show together with individual Western blots. Blots were blocked with 5% milk for 1 h, 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 are incubated with Pierce ECL from Thermo Fisher Scientific (cat. number 32106) prior to detection with HyBlot CL autoradiography films from Denville (cat. number 1159T41).

Antibody screening by immunoprecipitation
Immunoprecipitation was performed as described in our standard operating procedure. 14 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) or protein G-(for mouse antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Tubes were rocked~2 hours at 4°C followed by several 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) from Thermo Fisher Scientific (cat. number 87788), supplemented with protease inhibitor from MilliporeSigma (cat. number P8340). Lysates were rocked for 30 min at 4°C and spun at 110,000 x 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 a pre-cast mini 4-15% polyacrylamide gel. Prot-A:HRP (MilliporeSigma, cat. number P8651) was used as a secondary detection system at a dilution of 0.4 μg/mL. Refer to RRID recently added to the Antibody Registry (on January 2023), they will be available on the Registry website in coming weeks.
Antibody screening by immunofluorescence Immunofluorescence was performed as described in our standard operating procedure. 10 HAP1 WT and TMEM106B KO were labelled with CellTracker green (Thermo Fisher Scientific, cat. number C2925) or CellTracker deep red (Thermo Fisher Scientific, cat. number C34565) fluorescence dye, respectively. The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. number D3571) fluorescent stain. WT and KO cells were plated in 96 well glass plates (Perkin Elmer, cat. number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37 o C, 5% CO 2 . Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10mL) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL) for 15 min at room temperature and then washed three times with PBS. Cells were permeabilized in PBS with 0.1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature and blocked with PBS containing 5% BSA, 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0.01% Triton X-100) containing the primary TMEM106B antibodies overnight at 4°C. Cells were then washed 3 Â 10 min with IF buffer and incubated with the corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/mL for 1 hr at room temperature with DAPI. Cells were washed 3 Â 10 min with IF buffer and once with PBS.
Images were acquired on an ImageXpress micro widefield high-content microscopy system (Molecular Devices), using a 20x/0.45 NA air objective lens and scientific CMOS camera (16-

MINOR CONCERNS:
In their introduction, the authors speak of "a 135 amino acid portion of the protein from its luminal C-terminal domain", that might play a pathophysiological role.
Given this information, it might be interesting which of the six antibodies have epitopes in that specific domain. I there any information available? Some ideas to address this questions methodologically ? 1.
Results and discussion: "For Western blot experiments, we resolved proteins from WT and TMEM106B KO cell extracts and probed them side-by side with all antibodies in parallel ( Figure 1)." What means "side-by side" here? Do they come from the same membrane in order to avoid membrane-to-membrane variation? In the best case, in future experiments, the authors could even considering cutting the lanes vertically in order to even avoid lane-to-lane variation (cf. Pubmed ID 30768763).
2. Figure 1, panel PA5-34353: I am a bit surprised that the "scratches" and inhomogenities in protein transfer is not equally visible in the total protein staining (ponceau). I would kindly ask the authors to check in their data files that the ponceau staining is really originating from the same membrane, because the loading control would be invalid otherwise.
3.   Concluding sentence in the results section: "we have screened many TMEM106B commercial antibodies" I recommend avoiding vague words like "many" where a more concrete indication ("six") is possible. Same for the word "several" in the same sentence.

7.
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