The identification of high-performing antibodies for Charged multivesicular body protein 2b for use in Western Blot, immunoprecipitation and immunofluorescence

Charged multivesicular body protein 2B is a subunit of the endosomal sorting complex required for transport III (ESRCT-III), a complex implicated in the lysosomal degradation pathway and formation of multivesicular bodies. Mutations to the CHMP2B gene can result in abnormal protein aggregates in neurons and is therefore predicted to be associated in neurodegenerative diseases, including across the ALS-FTD spectrum. Through our standardized experimental protocol which compares read-outs in knockout cell lines and isogenic parental controls, this study aims to enhance the reproducibility of research on this target by characterizing eight commercial antibodies against charged multivesicular body protein 2b using Western Blot, immunoprecipitation, and immunofluorescence. 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
Charged multivesicular body protein 2B, encoded by the CHMP2B gene, is a core component of the endosomal sorting complex required for transport III (ESCTR-III) which plays a pivotal role in the biogenesis of multivesicular bodies (MVB) and is thus involved in endocytic trafficking of proteins. 1 MVB's are late endosomes formed by scission of intraluminal vesicles from the limiting membrane of the endosome to then deliver cargo proteins to the lysosome, enabling degradation of membrane proteins. 2,3 As a subunit of the ESCRT-III complex, Charged multivesicular body protein 2 is essential to the pathway of lysosomal degradation.
Mutations to the CHMP2B gene have been predicted to be associated with amyotrophic lateral sclerosis (ALS) 4 and frontotemporal dementia (FTD). 5 Affected neurons having abnormal ubiquitin-positive protein deposits which can be attributed to dysfunctional lysosomal degradation. 1 CHMP2B mutations related to the ALS-FTD spectrum have advanced the understanding of the role endosomallysosomal and autophagic dysregulation play in neurodegeneration. 1 As the exact mechanisms remain unknown, the availability of high-quality Charged multivesicular body protein 2 antibodies would greatly facilitate mechanistic studies.
Here, we compared the performance of a range of commercially available antibodies for Charged multivesicular body protein 2b and identified high-performing antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of Charged multivesicular body protein 2 properties and function.

Results and discussion
Our standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells. 6,7 To identify a cell line that expresses adequate levels of Charged multivesicular body protein 2b protein to provide sufficient signal to noise, we examined public proteomics databases, namely PaxDB 8 and DepMap. 9 U2OS was identified as a suitable cell line and thus U2OS was modified with CRISPR/Cas9 to knockout the corresponding CHMP2B gene (Table 1).
In conclusion, we have screened Charged multivesicular body protein 2b commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies under our standardized experimental conditions. The underlying data can be found on Zenodo. 21,22 Methods Antibodies All Charged multivesicular body protein 2b antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly. 23 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). Antibody screening by immunoprecipitation Immunoprecipitation was performed as described in our standard operating procedure. 26 Antibody-bead conjugates were prepared by adding 1 μg or 2 μL of antibody at an unknown concentration to 500 μL of Pierce IP Lysis Buffer When the concentration was not indicated by the supplier, which was the case for antibody MA5-21591*, the antibody was tested at 1/1000. Antibody dilution used: MAB7509* at 1/400; MA5-21591* at 1/1000; MA5-36184** at 1/500; ab157208** at 1/1000; 76173** at 1/1000; GTX118181 at 1/1000; GTX109610 at 1/1000; A13410 at 1/500. Predicted band size: 24 kDa. *Monoclonal antibody; **Recombinant antibody.
(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) (Thermo Fisher Scientific, cat. number 10002D and 10004D, respectively). Pierce IP Lysis Buffer was supplemented with the Halt Protease Inhibitor Cocktail 100X (Thermo Fisher Scientific, cat. number 78446) at a final concentration of 1Â. Tubes were rocked for~2 hrs at 4°C followed by several washes to remove unbound antibodies.
U2OS 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) supplemented with protease inhibitor. Lysates were rocked for 30 min at 4°C and spun at 110,000 Â 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 hours 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 4-20% 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 immunoblot. Antibody screening by immunofluorescence Immunofluorescence was performed as described in our standard operating procedure. 7,10-20 U2OS WT and CHMP2B KO were labelled with a green and a far-red fluorescence dye, respectively (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 37 o C, 5% CO. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10 ml) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL). 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 Charged multivesicular body protein 2b antibodies overnight at 4°C. Cells were then washed 3 Â 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 Â 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 63Â oil objective (NA=1.40). Analysis was done using the Zen navigation software (Zeiss). All cell images represent a single focal plane.  Exceptions were given to antibodies ab157208** and A13410, which were titrated to 1/1000 and 1/800, respectively, as the signals were too weak when following the suppliers' recommendations. When the concentration was not indicated by the supplier, which was the case for antibodies MA5-21791* and 76173*, we tested antibodies at 1/500. At this concentration, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: MAB7509* at 1/500; MA5-21591* at 1/500; MA5-36184** at 1/1000; ab157208** at 1/100; 76173** at 1/500; GTX118181 at 1/500; GTX109610 at 1/1000; A13410 at 1/800. Bars=10 μm. *Monoclonal antibody; **Recombinant antibody. This work is extremely helpful for researchers looking to source the best commercially available antibody to investigate their protein of interest, in particular the use of KO lines as a control to fully validate the specificity of the bands seen on the western blot images is powerful. The inclusion of the ponceau membranes to show protein transfer, and the inclusion of the whole length of the blot (to show non-specific bands) is a thorough and informative presentation of the antibody validation.
For the immunofluorescence data, the mosaic plating of the cells to allow the direct comparison of staining in adjacent cells allows for direct comparison and removes any variability due to differences in the staining protocol between conditions. This makes the comparisons between cells extremely powerful within images.
I could suggest two additions to the paper that would be interesting for researchers looking at CHMP2B and would add to the general usefulness of this antibody insight for the research community.
CHMP2B is generally cytosolic, and is temporarily recruited to the ESCRT-III complex to facilitate membrane invagination prior to VPS4 recruitment which then allows for membrane scission and ESCRT-III disassembly. When VPS4 is not fusion competent, CHMP2B is held in punctate structures in the cell 1 . In the immunofluorescence images in this article, the CHMP2B looks highly cytosolic as expected, however the adjacent KO cells don't look particularly different to the WT cells. It is hard to see what is specific staining over background noise. To make this immunofluorescence antibody validation useful for the research community, inducing the punctate localisation of CHMP2B would be useful, in order to show true antibody specificity.
I would also suggest adding the recognition sequences for the antibodies. This would be helpful in the case of mutations in CHMP2B which are associated with frontotemporal dementia and amyotrophic lateral sclerosis 2 . In particular, the mutation of CHMP2B associated with a familial form of frontotemporal dementia results in a C terminally truncated form of the protein 3 . If the antibody recognition sequence resides within the truncated portion of the protein (as we have unfortunately encountered in the past with CHMP2B antibodies), then the usefulness of an otherwise "good" CHMP2B antibody is compromised. Thus this information would be helpful for the research community interested in CHMP2B in neurodegenerative disease.
In summary however, this paper is a powerful resource for any researchers looking to work with wildtype CHMP2B. The detailed methods (with antibody dilutions and table of research resource identifiers) and in particular the powerful direct comparisons of WT and KO by western blotting provide high confidence in commercial antibody choice for a researcher looking to select an antibody for CHMP2B research.