Keywords
Uniprot ID Q9UHD9, UBQLN2, Ubiquilin-2, antibody characterization, antibody validation, Western Blot, immunoprecipitation, immunofluorescence
This article is included in the YCharOS (Antibody Characterization through Open Science) gateway.
This article is included in the Cell & Molecular Biology gateway.
Uniprot ID Q9UHD9, UBQLN2, Ubiquilin-2, antibody characterization, antibody validation, Western Blot, immunoprecipitation, immunofluorescence
Ubiquilin-2, a protein encoded by the UBQLN2 gene, plays a critical role in protein degradation pathways; including the ubiquitin-proteasome system (UPS), autophagy and the endoplasmic reticulum-associated protein degradation (ERAD) pathway.1
Disease-causing variants of UBQLN2 have been identified in patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD).2 These UBQLN2 mutations are predicted to be acting on ALS pathological mechanisms by causing UPS and autophagy dysfunction,3 neuroinflammation4,5 and/or formation of stress granules.6,7 Mechanistic 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 Ubiquilin-2 and validated high-quality antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of Ubiquilin-2 properties and function.
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 Ubiquilin-2 to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express UBQLN2 at levels greater than 2.5 log2 (transcripts per million “TPM” +1), which we had found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available HAP1 cells expressed the UBQLN2 transcript at RNA levels above the average range of cancer cells analyzed. The parental and UBQLN2 KO HAP1 cells were obtained from Horizon Discovery (Table 1).
Institution | Catalog number | RRID (Cellosaurus) | Cell line | Genotype |
---|---|---|---|---|
Horizon Discovery | C631 | CVCL_Y019 | HAP1 | WT |
Horizon Discovery | HZGHC004089c001 | CVCL_TW09 | HAP1 | UBQLN2 KO |
For Western Blot experiments, we resolved proteins from WT and UBQLN2 KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1).
For immunoprecipitation experiments, we used each of the antibodies to immunopurify Ubiquilin-2 from HAP1 cell extracts. The performance of each antibody was evaluated by detecting the Ubiquilin-2 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).
For immunofluorescence experiments, 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 biases (Figure 3).
In conclusion, we have screened Ubiquilin-2 commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence and identified several high-performing antibodies under our standardized experimental conditions. The underlying data is uploaded to Zenodo.16,17
All Ubiquilin-2 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (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). Peroxidase-conjugated monoclonal anti-Flag M2 is from MilliporeSigma (cat. number A8592). Alexa-555-conjugated goat anti-mouse and anti-rabbit secondary antibodies are from Thermo Fisher Scientific (cat. number A21424 and A21429). The anti-FLAG (M2 clone) conjugated with Cy3 is from MilliporeSigma (cat. number A9594).
Company | Catalog number | Lot number | RRID (Antibody Registry/Addgene) | Clonality | Clone ID | Host | Concentration (μg/μl) | Vendors recommended applications |
---|---|---|---|---|---|---|---|---|
Abcam | ab190283* | GR3297673-4 | AB_2747782 | monoclonal | 6H9 | mouse | 1.00 | Wb, IF |
ABclonal | A9568 | 92790201 | AB_2772790 | polyclonal | - | rabbit | 2.42 | Wb, IF |
Aviva Systems Biology | ARP59353 | QC30343-40589 | AB_10865653 | polyclonal | - | rabbit | 0.50 | Wb |
Bio-techne | NBP2-25164* | 81919 | AB_2885154 | monoclonal | 6H9 | mouse | 1.00 | Wb, IF |
Cell Signaling Technology | 85509** | 1 | AB_2800056 | recombinant-mono | D7R2Z | rabbit | 0.12 | Wb, IP |
Developmental Studies Hybridoma Bank | PCRP-UBQLN2-1C7* | 2021-03-04 | AB_2619216 | monoclonal | PCRP-UBQLN2-1C7 | mouse | 0.03 | Wb, IP |
Proteintech | 23449-1-AP | 52217 | AB_2879282 | polyclonal | - | rabbit | 0.55 | Wb, IF |
Structural Genomics Consortium | Z-UBQLN2-7** | YSUBQLN2A-c001 | Addgene_166563 | recombinant-mono | YSUBQLN2A-c001 | human | 0.65 | IP |
Thermo Fisher Scientific | 35-4400* | WA320043 | AB_2533204 | monoclonal | 3B8A10 | mouse | 0.50 | Wb, IF |
Thermo Fisher Scientific | 37-7700* | VA300207 | AB_2533341 | monoclonal | 3D5E2 | mouse | 0.50 | Wb, IP, IF |
Both HAP1 WT and UBQLN2 KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.12 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 Blot experiments were performed as described in our standard operating procedure.13 HAP1 WT and UBQLN2 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 0089901), supplemented with 1× 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× g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and immunoblot. BLUelf prestained protein ladder from GeneDireX (cat. number PM008-0500) was used.
Western Blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific (cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer from bio-Rad (cat. number 1610772), loaded in Laemmli loading sample buffer from Thermo Fisher Scientific (cat. number AAJ61337AD) 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 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 iBright™ CL1500 Imaging System (Thermo Fisher Scientific, cat. number A44240).
Immunoprecipitation experiments were performed as described in our standard operating procedure.14 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 30μl of Dynabeads protein A- (for rabbit antibodies) or protein G- (for mouse antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively) or anti-Flag M2 magnetic beads from MilliporeSigma (cat. number M8823). Tubes were rocked for ~1 hr 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 30 min at 4°C and spun at 110,000× 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 ~1 hr at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of IP buffer and processed for SDS-PAGE and Western Blot on precast midi 4-20% Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific, cat number WXP42012BOX).
Immunofluorescence was performed as described in our standard operating procedure.10 HAP1 WT and UBQLN2 KO were labelled with CellTrackerTM green (Thermo Fisher Scientific, cat. number C2925) or CellTrackerTM 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 37oC, 5% CO2. 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 3 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 Ubiquilin-2 antibodies overnight at 4°C. Cells were then washed 3 × 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 × 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 20×/0.45 NA air objective lens and scientific CMOS camera (16-bit, 1.97mm field of view), equipped with 395, 475, 555 and 635 nm solid state LED lights (Lumencor Aura III light engine) and bandpass emission filters (432/36 nm, 520/35 nm, 600/37 nm and 692/40 nm) to excite and capture fluorescence emission for DAPI, CellTrackerTM Green, Alexa fluor 555 and CellTrackerTM Red, respectively. Images had pixel sizes of 0.68 × 0.68 microns. Exposure time was set with maximal (relevant) pixel intensity ~80% of dynamic range and verified on multiple wells before acquisition. Since the IF staining varied depending on the primary antibody used, the exposure time was set using the most intensely stained well as reference. Frequently, the focal plane varied slightly within a single field of view. To remedy this issue, a stack of three images per channel was acquired at a z-interval of 4 microns per field and best focus projections were generated during the acquisition (MetaExpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTrackerTM channels using CellPose v1.0 on green (WT) and far-red (KO) channels, using as parameters the ‘cyto’ model to detect whole cells, and using an estimated diameter tested for each cell type, between 15 and 20 microns.15 Masks were used to generate cell outlines for intensity quantificationFigures were assembled with Adobe Illustrator.
Zenodo: Antibody Characterization Report for Ubiquilin-2, https://doi.org/10.5281/zenodo.7459541. 16
Zenodo: Dataset for the Ubiquilin-2 antibody screening study, https://doi.org/10.5281/zenodo.7671135. 17
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. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below.
NeuroSGC/YCharOS/EDDU collaborative group: Riham Ayoubi, Thomas M. Durcan, Aled M. Edwards, Carl Laflamme, Ian McDowell, Peter S. McPherson, Chetan Raina, Wolfgang Reintsch and Kathleen Southern.
ABIF consortium: Claire M. Brown and Joel Ryan.
An earlier version of this of this article can be found on Zenodo (doi: 10.5281/zenodo.7459541).
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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: biophysics, protein structure and function, biomolecular condensates, protein quality control, and ubiquitin 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: Neurodegenerative disorders
Alongside their report, reviewers assign a status to the article:
Invited Reviewers | ||
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Version 1 30 Mar 23 |
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