Keywords
Uniprot ID Q8WYQ3, CHCHD10, Coiled-coil-helix-coiled-coil-helix domain-containing protein 10, antibody characterization, antibody validation, Western Blot, immunoprecipitation, immunofluorescence
This article is included in the Cell & Molecular Biology gateway.
This article is included in the YCharOS (Antibody Characterization through Open Science) gateway.
Uniprot ID Q8WYQ3, CHCHD10, Coiled-coil-helix-coiled-coil-helix domain-containing protein 10, antibody characterization, antibody validation, Western Blot, immunoprecipitation, immunofluorescence
The newly submitted version of the article clarifies in the introduction that CHCHD10 protein contains a single CHCH domain formed by two CX9C motifs.
See the authors' detailed response to the review by Derek P. Narendra
Coiled-coil-helix-coiled-coil-helix domain containing protein 10 (CHCHD10) is a protein localized to the mitochondrial intermembrane space, and is postulated to be involved in the maintenance of mitochondrial organization and cristae structure.1 Twin CX9C motifs make up the single coiled-coil-helix-coiled-coil-helix domain, which stabilizes the helix-turn-helix fold.2
Recent studies have demonstrated that CHCHD10 is important for neuronal health.1 As such, CHCHD10 gene variants have been reported in patients with ALS, FTD, Parkinson’s disease, motor neuron disease, and mitochondrial myopathy, suggesting that they contribute to neurodegenerative disease progression.1,3,4 Additional work is needed to understand the underlying function and regulation of CHCHD10, in its native and mutant conformation, to advance the development of therapeutic strategies for targeting these deteriorating diseases. Mechanistic studies would be greatly facilitated with the availability of high-quality CHCHD10 antibodies.
Here, we compared the performance of a range of commercially available CHCHD10 antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of CHCHD10 properties and function.
Our standard protocol involves comparing readouts from wild-type and knockout cells.5–9 The first step was to identify a cell line(s) that expresses sufficient levels of CHCHD10 to generate a measurable signal to noise. To this end, we examined the DepMap transcriptomics databases 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 (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available HAP1 cells expressed the CHCHD10 transcript at RNA levels above the average range of cancer cells analyzed. Parental and CHCHD10 knockout HAP1 cells were obtained from Horizon Discovery. Parental HCT116 cells were obtained from Abcam for immunoprecipitation experiments (Table 1).
Institution | Catalog number | RRID (Cellosaurus) | Cell line | Genotype |
---|---|---|---|---|
Horizon Discovery | C631 | CVCL_Y019 | HAP1 | WT |
Horizon Discovery | HZGHC005043c003 | CVCL_SI77 | HAP1 | CHCHD10 KO |
Abcam | ab255451 | CVCL_0291 | HCT116 | WT |
For Western Blot experiments, we resolved proteins from WT and CHCHD10 KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1).6–12
For immunoprecipitation experiments, we used the antibodies to immunopurify CHCHD10 from cell extracts. The performance of each antibody was evaluated using Western Blot by detecting the CHCHD10 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).6–12
For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy.13 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 CHCHD10 commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence and identified high-quality antibodies under our standardized experimental conditions. The underlying data can be found on Zenodo.14,15
All CHCHD10 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (RRID), to ensure the antibodies are cited properly.16 Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424).
Company | Catalog number | Lot number | RRID (Antibody Registry) | Clonality | Clone ID | Host | Concentration (μg/μL) | Vendors recommended applications |
---|---|---|---|---|---|---|---|---|
Proteintech | 25671-1-AP | 53318 | AB_2880187 | polyclonal | - | rabbit | 0.30 | Wb, IP, IF |
Thermo Fisher Scientific | MA5-27532* | VL3152361A | AB_2724131 | monoclonal | OTI2B6 | mouse | 1.00 | Wb |
Thermo Fisher Scientific | MA5-27535* | VL3152362A | AB_2724132 | monoclonal | OTI3B8 | mouse | 1.00 | Wb |
Thermo Fisher Scientific | MA5-27531* | VL3152369 | AB_2724133 | monoclonal | OTI4C12 | mouse | 1.00 | Wb |
Both HAP1 WT and CHCHD10 KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.17 Cells were cultured in DMEM high-glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent cat. number 609065), 100 IU penicillin and 100 μg/mL streptomycin (Wisent cat. number 450201).
Western Blots were performed as described in our standard operating procedure.18 HAP1 WT and CHCHD10 KO were collected in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1.0 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 1x protease inhibitor cocktail mix (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 large 8-16% 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 Blot. 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 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 Pierce ECL from Thermo Fisher Scientific (cat. number 32106) prior to detection with the HyBlot CL autoradiography films from Denville (cat. number 1159T41).
Immunoprecipitation was performed as described in our standard operating procedure.19 Antibody-bead conjugates were prepared by adding 1.0 μg of antibody to 500 μL of phosphate-buffered saline (PBS) (Wisent, cat. number 311-010-CL) with 0,01% triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) in a 1.5 mL microcentrifuge tube, together with 30 μL of protein A- (for rabbit antibodies) or protein G- (for mouse antibodies) Sepharose beads. Tubes were rocked overnight at 4°C followed by two washes to remove unbound antibodies.
HCT116 WT were collected in HEPES buffer (20 mM HEPES, 100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor. Lysates were rocked for 30 min at 4°C and spun at 110,000 x 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 HEPES lysis buffer and processed for SDS-PAGE and Western Blot on 8-16% polyacrylamide gels. As secondary detections systems, the Veriblot for immunoprecipitation detection reagent and the anti-mouse IgG for immunoprecipitation (HRP) from Abcam (cat. number ab131366 and ab131368, respectively) were used.
Immunofluorescence was performed as described in our standard operating procedure.6–13 HAP1 WT and CHCHD10 KO were labelled with a green and a far-red fluorescence dye, respectively. The fluorescent dyes used are from Thermo Fisher Scientific (cat. number C2925 and C34565). The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. Number D3571) fluorescent stain. WT and KO cells were plated on glass coverslips as a mosaic and incubated for 24 hrs in a cell culture incubator at 37oC, 5% CO2. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10ml) in PBS for 15 min at room temperature and then washed 3 times with PBS. Cells were permeabilized in PBS with 0,1% Triton X-100 for 10 min at room temperature and blocked with PBS with 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 CHCHD10 antibodies overnight at 4°C. Cells were then washed 3 × 10 min with IF buffer and incubated with 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 × 10 min with IF buffer and once with PBS. Coverslips were mounted on a microscopic slide using fluorescence mounting media (DAKO).
Imaging was performed using a Zeiss LSM 880 laser scanning confocal microscope equipped with a Plan-Apo 40x oil objective (NA = 1.40). Analysis was done using the Zen navigation software (Zeiss). 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).
Zenodo: Antibody Characterization Report for CHCHD10, https://doi.org/10.5281/zenodo.5259992. 14
Zenodo: Dataset for the CHCHD10 antibody screening study, https://doi.org/10.5281/zenodo.7779321. 15
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).
We would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources and collaboration. Members of the group can be found below.
NeuroSGC/YCharOS/EDDU collaborative group: Riham Ayoubi, Aled M. Edwards, Carl Laflamme, Peter S. McPherson, Chetan Raina, and Kathleen Southern
An earlier version of this of this article can be found on Zenodo (doi: 10.5281/zenodo.5259992)
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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: Frontotemporal dementia, lipid metabolism, antisense oligonucleotides
References
1. Burstein SR, Valsecchi F, Kawamata H, Bourens M, et al.: In vitro and in vivo studies of the ALS-FTLD protein CHCHD10 reveal novel mitochondrial topology and protein interactions.Hum Mol Genet. 2018; 27 (1): 160-177 PubMed Abstract | Publisher Full TextCompeting Interests: No competing interests were disclosed.
Reviewer Expertise: neurogenetics, mitochondrial biology
Is the rationale for creating the dataset(s) clearly described?
Partly
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
References
1. Anderson CJ, Bredvik K, Burstein SR, Davis C, et al.: ALS/FTD mutant CHCHD10 mice reveal a tissue-specific toxic gain-of-function and mitochondrial stress response.Acta Neuropathol. 2019; 138 (1): 103-121 PubMed Abstract | Publisher Full TextCompeting Interests: No competing interests were disclosed.
Reviewer Expertise: neurogenetics, mitochondrial biology
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: Molecular mechanisms for infrared sensing in snakes and function & potential medical applications of snake venom.
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