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

Walaa Alshafie; Maryam Fotouhi; Riham Ayoubi; Irina Shlaifer; Kathleen Southern; Peter S. McPherson; Carl Laflamme; NeuroSGC/YCharOS/EDDU collaborative group

doi:10.12688/f1000research.139755.1

Home Browse The identification of high-performing antibodies for Charged multivesicular...

ALL Metrics

-

Views

-

Downloads

Get PDF

Get XML

Export

▬

✚

Data Note

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

[version 1; peer review: 2 approved]

Walaa Alshafie¹, Maryam Fotouhi¹, Riham Ayoubi¹, [...] Irina Shlaifer², Kathleen Southern¹, Peter S. McPherson¹, Carl Laflamme ¹, NeuroSGC/YCharOS/EDDU collaborative group

Walaa Alshafie¹, Maryam Fotouhi¹, [...] Riham Ayoubi¹, Irina Shlaifer², Kathleen Southern¹, Peter S. McPherson¹, Carl Laflamme ¹, NeuroSGC/YCharOS/EDDU collaborative group

PUBLISHED 25 Jul 2023

Author details Author details

¹ Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada
² The Neuro’s Early Drug Discovery Unit (EDDU), Structural Genomics Consortium, McGill University, Montreal, Québec, H3A 2B4, Canada

Walaa Alshafie
Roles: Investigation, Methodology, Validation

Maryam Fotouhi
Roles: Investigation, Validation

Riham Ayoubi
Roles: Investigation, Methodology, Validation, Visualization, Writing – Review & Editing

Irina Shlaifer
Roles: Investigation, Methodology

Kathleen Southern
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Peter S. McPherson
Roles: Conceptualization, Data Curation, Funding Acquisition, Resources, Supervision

Carl Laflamme
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Resources, Supervision, Validation, Visualization

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Cell & Molecular Biology gateway.

This article is included in the YCharOS (Antibody Characterization through Open Science) gateway.

Abstract

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.

Keywords

Uniprot ID Q9UQN3, CHMP2B, Charged multivesicular body protein 2b, antibody characterization, antibody validation, Western Blot, immunoprecipitation, immunofluorescence

Corresponding author: Carl Laflamme

Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam-Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific.

Grant information: This work was supported in part by the ALS-Reproducible Antibody Platform (ALS-RAP). ALS-RAP is a private-public partnership created by the ALS Association (USA), the Motor Neurone Disease Association (UK), and the ALS Society of Canada. The grant was from a Canadian Institutes of Health Research Foundation Grant (FDN154305) and by the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (OGI-210). RA and WA were supported by a Mitacs fellowship.  
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Alshafie W et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Alshafie W, Fotouhi M, Ayoubi R et al. The identification of high-performing antibodies for Charged multivesicular body protein 2b for use in Western Blot, immunoprecipitation and immunofluorescence [version 1; peer review: 2 approved]. F1000Research 2023, 12:884 (https://doi.org/10.12688/f1000research.139755.1) First published: 25 Jul 2023, 12:884 (https://doi.org/10.12688/f1000research.139755.1) Latest published: 25 Jul 2023, 12:884 (https://doi.org/10.12688/f1000research.139755.1)

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 endosomal-lysosomal 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 PaxDB⁸ 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).

Table 1. Summary of the cell lines used.

Institution	Catalog number	RRID (Cellosaurus)	Cell line	Genotype
ATCC	HTB-96	CVCL_0042	U2OS	WT
Montreal Neurological Institute	-	CVCL_B6JX	U2OS	CHMP2B KO

For Western Blot experiments, we resolved proteins from WT and CHMP2B KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1).⁷^,¹⁰^–¹⁹

Figure 1. Charged multivesicular body protein 2b antibody screening by Western Blot.

Lysates of U2OS (WT and CHMP2B KO) were prepared and 50 μg of protein were processed for Western Blot with the indicated Charged multivesicular body protein 2b antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. 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.

For immunoprecipitation experiments, we used the antibodies to immunopurify Charged multivesicular body protein 2b from U2OS cell extracts. The performance of each antibody was evaluated by detecting the Charged multivesicular body protein 2b protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).⁷^,¹⁰^–¹⁹

Figure 2. Charged multivesicular body protein 2b antibody screening by immunoprecipitation.

U2OS lysates were prepared, and IP was performed using 1.0 μg of the indicated Charged multivesicular body protein 2b antibodies pre-coupled to Dynabeads protein G or protein A. Samples were washed and processed for Western Blot with the indicated Charged multivesicular body protein 2b antibody. For Western Blot, ab157208** was used at 1/2000. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitated. *Monoclonal antibody; **Recombinant antibody.

For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy.²⁰ 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).

Figure 3. Charged multivesicular body protein 2b antibody screening by immunofluorescence.

U2OS WT and CHMP2B 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 on coverslips. Cells were stained with the indicated Charged multivesicular body protein 2b antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with yellow and magenta dashed line, respectively. Antibody dilutions were chosen according to the recommendations of the antibody supplier. 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.

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).

Table 2. Summary of the Charged multivesicular body protein 2b antibodies tested.

Company	Catalog number	Lot number	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μl)	Vendors recommended applications
Bio-Techne	MAB7509*	CHEB0112101	AB_2885148	monoclonal	791521	mouse	0.50	Wb
Thermo Fisher Scientific	MA5-21591*	WA3152391	AB_2576481	monoclonal	2H6-1E6	mouse	0.50	Other application
Thermo Fisher Scientific	MA5-36184**	VL3152619	AB_2890433	recombinant-mono	JE54-35	rabbit	1.00	Wb, IF
Abcam	ab157208**	GR117930-3	AB_2885096	recombinant-mono	EPR10807(B)	rabbit	0.13	Wb, IP, IF
Cell Signaling Technology	76173**	1	AB_2799880	recombinant-mono	D4G3K	rabbit	not provided	Wb, IP
GeneTex	GTX118181	40625	AB_11174469	polyclonal	-	rabbit	0.59	Wb, IF
GeneTex	GTX109610	40681	AB_11163162	polyclonal	-	rabbit	1.00	Wb, IF
ABclonal	A13410	13540101	AB_2760272	polyclonal	-	rabbit	0.85	Wb, IF

* Monoclonal antibody.

** Recombinant antibody.

CRISPR/Cas9 genome editing

Cell lines used are listed in Table 1. U2OS CHMP2B KO clone was generated with low passage cells using an open-access protocol available on Zenodo.org. The sequence of the guide RNA is the following: CCAAACAACUUGUGCAUCUA.

Cell culture

Both U2OS WT and CHMP2B KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.²⁴ 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).

Antibody screening by Western Blot

Western Blots were performed as described in our standard operating procedure.²⁵ U2OS WT and CHMP2B 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 89901) supplemented with 1× protease inhibitor cocktail mix (MilliporeSigma, cat. number P8340). 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 Western Blot. BLUelf prestained protein ladder (GeneDireX, cat. number PM008-0500) was used.

Western Blots were performed with large 4-20% 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 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 HyBlot CL autoradiography films (Denville, cat. number 1159T41).

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 (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 1×. 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 × 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^oC, 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 × 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 63× 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).

Data availability

Underlying data

Zenodo: Antibody Characterization Report for Charged multivesicular body protein 2b, https://doi.org/10.5281/zenodo.6370501.²¹

Zenodo: Dataset for the Charged multivesicular body protein 2b antibody screening study, https://doi.org/10.5281/zenodo.8139356.²²

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgments

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, Thomas M. Durcan, Aled M. Edwards, Carl Laflamme, Peter S. McPherson, Chetan Raina and Kathleen Southern.

Thank you to the Structural Genomics Consortium, a registered charity (no. 1097737), for your support on this project. The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no. OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.

An earlier version of this of this article can be found on Zenodo (doi: 10.5281/zenodo.6370501).

References

1. Ugbode C, West RJH: Lessons learned from CHMP2B, implications for frontotemporal dementia and amyotrophic lateral sclerosis. Neurobiol. Dis. 2021; 147: 105144. Publisher Full Text
2. Li X, Bao H, Wang Z, et al.: Biogenesis and Function of Multivesicular Bodies in Plant Immunity. Front. Plant Sci. 2018; 9: 979. PubMed Abstract | Publisher Full Text | Free Full Text
3. Piper RC, Katzmann DJ: Biogenesis and function of multivesicular bodies. Annu. Rev. Cell Dev. Biol. 2007; 23: 519–547. PubMed Abstract | Publisher Full Text | Free Full Text
4. Parkinson N, Ince PG, Smith MO, et al.: ALS phenotypes with mutations in CHMP2B (charged multivesicular body protein 2B). Neurology. 2006; 67(6): 1074–1077. PubMed Abstract | Publisher Full Text
5. Skibinski G, Parkinson NJ, Brown JM, et al.: Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nat. Genet. 2005; 37(8): 806–808. PubMed Abstract | Publisher Full Text
6. Laflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8: 8. Publisher Full Text
7. Alshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text
8. Wang M, Herrmann CJ, Simonovic M, et al.: Version 4.0 of PaxDb: Protein abundance data, integrated across model organisms, tissues, and cell-lines. Proteomics. 2015; 15(18): 3163–3168. PubMed Abstract | Publisher Full Text | Free Full Text
9. DepMap, Broad: DepMap 19Q3 Public ed.2019.
10. Alshafie W, Ayoubi R, Fotouhi M, et al.: The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 172. Publisher Full Text
11. Ayoubi R, Fotouhi M, Southern K, et al.: The identification of high-performing antibodies for transmembrane protein 106B (TMEM106B) for use in Western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 308. Publisher Full Text
12. Ayoubi R, Alshafie W, Shlaifer I, et al.: The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 324. Publisher Full Text
13. Ayoubi R, McDowell I, Fotouhi M, et al.: The identification of high-performing antibodies for Profilin-1 for use in Western blot, immunoprecipitation and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 348. Publisher Full Text
14. McDowell I, Ayoubi R, Fotouhi M, et al.: The identification of high-preforming antibodies for Ubiquilin-2 for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 355. Publisher Full Text
15. Alshafie W, Fotouhi M, Ayoubi R, et al.: The identification of high-performing antibodies for RNA-binding protein FUS for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 1 approved with reservations]. F1000Res. 2023; 12: 376. PubMed Abstract | Publisher Full Text | Free Full Text
16. Ayoubi R, Alshafie W, Southern K, et al.: The identification of high-performing antibodies for Coiled-coil-helix-coiled-coil-helix domain containing protein 10 (CHCHD10) for use in Western Blot, immunoprecipitation and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 403. Publisher Full Text
17. Worrall D, Ayoubi R, Fotouhi M, et al.: The identification of high-performing antibodies for TDP-43 for use in Western Blot, immunoprecipitation and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 277. PubMed Abstract | Publisher Full Text | Free Full Text
18. Ayoubi R, Fotouhi M, Southern K, et al.: The identification of high-performing antibodies for Vacuolar protein sorting-associated protein 35 (hVPS35) for use in Western Blot, immunoprecipitation and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 452. Publisher Full Text
19. Ayoubi R, Alshafie W, You Z, et al.: The identification of high-performing antibodies for Superoxide dismutase [Cu-Zn] 1 (SOD1) for use in Western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 391. Publisher Full Text
20. Alshafie W, McPherson P, Laflamme C: Antibody screening by Immunofluorescence. Zenodo. 2021. Publisher Full Text
21. Fotouhi M, Alshafie W, Shlaifer I, et al.: Antibody Characterization Report for Charged multivesicular body protein 2b. [Dataset]. Zenodo. 2022. Publisher Full Text
22. Southern K: Dataset for the Charged multivesicular body protein 2b antibody screening study [Dataset]. Zenodo. 2023. Publisher Full Text
23. Bandrowski A, Pairish M, Eckmann P, et al.: The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2022; 51: D358–D367. Publisher Full Text
24. Bairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29(2): 25–38. PubMed Abstract | Publisher Full Text | Free Full Text
25. Ayoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot. Zenodo. 2021. Publisher Full Text
26. Ayoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation. Zenodo. 2021. Publisher Full Text

Comments on this article Comments (0)

Version 1

VERSION 1 PUBLISHED 25 Jul 2023

Author details Author details

¹ Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada
² The Neuro’s Early Drug Discovery Unit (EDDU), Structural Genomics Consortium, McGill University, Montreal, Québec, H3A 2B4, Canada

Walaa Alshafie
Roles: Investigation, Methodology, Validation

Maryam Fotouhi
Roles: Investigation, Validation

Riham Ayoubi
Roles: Investigation, Methodology, Validation, Visualization, Writing – Review & Editing

Irina Shlaifer
Roles: Investigation, Methodology

Kathleen Southern
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Peter S. McPherson
Roles: Conceptualization, Data Curation, Funding Acquisition, Resources, Supervision

Carl Laflamme
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Resources, Supervision, Validation, Visualization

Competing interests

For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam-Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific.

Grant information

This work was supported in part by the ALS-Reproducible Antibody Platform (ALS-RAP). ALS-RAP is a private-public partnership created by the ALS Association (USA), the Motor Neurone Disease Association (UK), and the ALS Society of Canada. The grant was from a Canadian Institutes of Health Research Foundation Grant (FDN154305) and by the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (OGI-210). RA and WA were supported by a Mitacs fellowship.  
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (1)

version 1

Published: 25 Jul 2023, 12:884

https://doi.org/10.12688/f1000research.139755.1

Copyright

© 2023 Alshafie W et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Download

Export To

metrics

	Views	Downloads
F1000Research	-	-
PubMed Central Data from PMC are received and updated monthly.	-	-

Citations

0

SEE MORE DETAILS

CITE

how to cite this article

Alshafie W, Fotouhi M, Ayoubi R et al. The identification of high-performing antibodies for Charged multivesicular body protein 2b for use in Western Blot, immunoprecipitation and immunofluorescence [version 1; peer review: 2 approved] F1000Research 2023, 12:884 (https://doi.org/10.12688/f1000research.139755.1)

NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

track

receive updates on this article

Track an article to receive email alerts on any updates to this article.

Open Peer Review

Current Reviewer Status: ?

Key to Reviewer Statuses VIEW HIDE

ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 1

VERSION 1

PUBLISHED 25 Jul 2023

Views

4

Reviewer Report 23 Aug 2023

Xiao-Xin Yan, Department of Anatomy and Neurobiology, Central South University Xiangya School of Medicine, Changsha, Hunan, China

Approved

https://doi.org/10.5256/f1000research.153058.r196057

This manuscript reports the characterization of CHMP2B antibodies for the use in Western Blot, immunoprecipitation and immunofluorescence. Good CHMP2B antibodies would be very useful for study of not only ALS-FTD disorders as the authors indicated, but also for other neurodegenerative ... Continue reading

This manuscript reports the characterization of CHMP2B antibodies for the use in Western Blot, immunoprecipitation and immunofluorescence. Good CHMP2B antibodies would be very useful for study of not only ALS-FTD disorders as the authors indicated, but also for other neurodegenerative diseases or conditions, such as aging and AD-related formation of granulovacuolar degeneration (GVD) (Jiang et al., 2022¹). The utility of the antibodies in immunoprecipitation is particularly informative for research into potential interplay between CHMP2B and other molecular partners in the neuroscience field. I would appreciate if the authors could extend some more relavence either in the introduction or discussion on CHMP2B in relevance to GVD formation or protein sorting in neurodegeneratrive disorders.

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

References

1. Jiang J, Yang C, Ai JQ, Zhang QL, et al.: Intraneuronal sortilin aggregation relative to granulovacuolar degeneration, tau pathogenesis and sorfra plaque formation in human hippocampal formation.Front Aging Neurosci. 2022; 14: 926904 PubMed Abstract | Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Neurodevelopment, brain aging, Alzheimer's disease

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

CITE

Report a concern

Respond or Comment

Views

7

Reviewer Report 08 Aug 2023

Emma L. Clayton, UK Dementia Research Institute, King's College London, London, UK

Approved

https://doi.org/10.5256/f1000research.153058.r190521

In line with the remit of the YCharOS (antibody characterisation through open science) initiative which seeks to characterise commercially available antibodies for every human protein, Alshafie et al. have compared the ability of a range of commercially available antibodies to detect ... Continue reading

In line with the remit of the YCharOS (antibody characterisation through open science) initiative which seeks to characterise commercially available antibodies for every human protein, Alshafie et al. have compared the ability of a range of commercially available antibodies to detect CHMP2B by western blotting and immunofluorescence. The model used for these validation experiments (U2OS) was selected as a line known to express adequate levels of CHMP2B to provide sufficient signal to noise. Antibody validation was complemented through use of CRISPR/Cas9 knockout to generate CHMP2B deficient control lines.

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 cell¹. 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 sclerosis². In particular, the mutation of CHMP2B associated with a familial form of frontotemporal dementia results in a C terminally truncated form of the protein³. 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.

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

References

1. Clayton E, Milioto C, Muralidharan B, Norona F, et al.: Frontotemporal dementia causative CHMP2B impairs neuronal endolysosomal traffic-rescue byTMEM106B knockdown. Brain. 2018; 141 (12): 3428-3442 Publisher Full Text
2. Ugbode C, West R: Lessons learned from CHMP2B, implications for frontotemporal dementia and amyotrophic lateral sclerosis. Neurobiology of Disease. 2021; 147. Publisher Full Text
3. Skibinski G, Parkinson N, Brown J, Chakrabarti L, et al.: Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nature Genetics. 2005; 37 (8): 806-808 Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Neurodegeneration, Membrane trafficking, Synaptopathy

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

CITE

Report a concern

Respond or Comment

Comments on this article Comments (0)

Version 1

VERSION 1 PUBLISHED 25 Jul 2023

Open Peer Review

Reviewer Status

Reviewer Reports

	Invited Reviewers
	1	2
Version 1 25 Jul 23	read	read

Emma L. Clayton, King's College London, London, UK
Xiao-Xin Yan, Central South University Xiangya School of Medicine, Changsha, Hunan, China

Comments on this article

All Comments(0)

Add a comment

Sign up for content alerts

Browse by related subjects

Back to all reports

Reviewer Report

4 Views

23 Aug 2023 | for Version 1

Xiao-Xin Yan, Department of Anatomy and Neurobiology, Central South University Xiangya School of Medicine, Changsha, Hunan, China

4 Views Cite this report Responses(0)

Approved

This manuscript reports the characterization of CHMP2B antibodies for the use in Western Blot, immunoprecipitation and immunofluorescence. Good CHMP2B antibodies would be very useful for study of not only ALS-FTD disorders as the authors indicated, but also for other neurodegenerative diseases or conditions, such as aging and AD-related formation of granulovacuolar degeneration (GVD) (Jiang et al., 2022¹). The utility of the antibodies in immunoprecipitation is particularly informative for research into potential interplay between CHMP2B and other molecular partners in the neuroscience field. I would appreciate if the authors could extend some more relavence either in the introduction or discussion on CHMP2B in relevance to GVD formation or protein sorting in neurodegeneratrive disorders.

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

References

1. Jiang J, Yang C, Ai JQ, Zhang QL, et al.: Intraneuronal sortilin aggregation relative to granulovacuolar degeneration, tau pathogenesis and sorfra plaque formation in human hippocampal formation.Front Aging Neurosci. 2022; 14: 926904 PubMed Abstract | Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Neurodevelopment, brain aging, Alzheimer's disease

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

7 Views

08 Aug 2023 | for Version 1

Emma L. Clayton, UK Dementia Research Institute, King's College London, London, UK

7 Views Cite this report Responses(0)

Approved

In line with the remit of the YCharOS (antibody characterisation through open science) initiative which seeks to characterise commercially available antibodies for every human protein, Alshafie et al. have compared the ability of a range of commercially available antibodies to detect CHMP2B by western blotting and immunofluorescence. The model used for these validation experiments (U2OS) was selected as a line known to express adequate levels of CHMP2B to provide sufficient signal to noise. Antibody validation was complemented through use of CRISPR/Cas9 knockout to generate CHMP2B deficient control lines.

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 cell¹. 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 sclerosis². In particular, the mutation of CHMP2B associated with a familial form of frontotemporal dementia results in a C terminally truncated form of the protein³. 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.

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

References

1. Clayton E, Milioto C, Muralidharan B, Norona F, et al.: Frontotemporal dementia causative CHMP2B impairs neuronal endolysosomal traffic-rescue byTMEM106B knockdown. Brain. 2018; 141 (12): 3428-3442 Publisher Full Text
2. Ugbode C, West R: Lessons learned from CHMP2B, implications for frontotemporal dementia and amyotrophic lateral sclerosis. Neurobiology of Disease. 2021; 147. Publisher Full Text
3. Skibinski G, Parkinson N, Brown J, Chakrabarti L, et al.: Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nature Genetics. 2005; 37 (8): 806-808 Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Neurodegeneration, Membrane trafficking, Synaptopathy

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

Responses (0)

[1] 1. Ugbode C, West RJH: Lessons learned from CHMP2B, implications for frontotemporal dementia and amyotrophic lateral sclerosis. Neurobiol. Dis. 2021; 147: 105144. Publisher Full Text

[2] 2. Li X, Bao H, Wang Z, et al.: Biogenesis and Function of Multivesicular Bodies in Plant Immunity. Front. Plant Sci. 2018; 9: 979. PubMed Abstract | Publisher Full Text | Free Full Text

[3] 3. Piper RC, Katzmann DJ: Biogenesis and function of multivesicular bodies. Annu. Rev. Cell Dev. Biol. 2007; 23: 519–547. PubMed Abstract | Publisher Full Text | Free Full Text

[4] 4. Parkinson N, Ince PG, Smith MO, et al.: ALS phenotypes with mutations in CHMP2B (charged multivesicular body protein 2B). Neurology. 2006; 67(6): 1074–1077. PubMed Abstract | Publisher Full Text

[5] 5. Skibinski G, Parkinson NJ, Brown JM, et al.: Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nat. Genet. 2005; 37(8): 806–808. PubMed Abstract | Publisher Full Text

[6] 6. Laflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8: 8. Publisher Full Text

[7] 7. Alshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text

[8] 8. Wang M, Herrmann CJ, Simonovic M, et al.: Version 4.0 of PaxDb: Protein abundance data, integrated across model organisms, tissues, and cell-lines. Proteomics. 2015; 15(18): 3163–3168. PubMed Abstract | Publisher Full Text | Free Full Text

[9] 9. DepMap, Broad: DepMap 19Q3 Public ed.2019.

[10] 10. Alshafie W, Ayoubi R, Fotouhi M, et al.: The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 172. Publisher Full Text

[11] 11. Ayoubi R, Fotouhi M, Southern K, et al.: The identification of high-performing antibodies for transmembrane protein 106B (TMEM106B) for use in Western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 308. Publisher Full Text

[12] 12. Ayoubi R, Alshafie W, Shlaifer I, et al.: The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 324. Publisher Full Text

[13] 13. Ayoubi R, McDowell I, Fotouhi M, et al.: The identification of high-performing antibodies for Profilin-1 for use in Western blot, immunoprecipitation and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 348. Publisher Full Text

[14] 14. McDowell I, Ayoubi R, Fotouhi M, et al.: The identification of high-preforming antibodies for Ubiquilin-2 for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 355. Publisher Full Text

[15] 15. Alshafie W, Fotouhi M, Ayoubi R, et al.: The identification of high-performing antibodies for RNA-binding protein FUS for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 1 approved with reservations]. F1000Res. 2023; 12: 376. PubMed Abstract | Publisher Full Text | Free Full Text

[16] 16. Ayoubi R, Alshafie W, Southern K, et al.: The identification of high-performing antibodies for Coiled-coil-helix-coiled-coil-helix domain containing protein 10 (CHCHD10) for use in Western Blot, immunoprecipitation and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 403. Publisher Full Text

[17] 17. Worrall D, Ayoubi R, Fotouhi M, et al.: The identification of high-performing antibodies for TDP-43 for use in Western Blot, immunoprecipitation and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 277. PubMed Abstract | Publisher Full Text | Free Full Text

[18] 18. Ayoubi R, Fotouhi M, Southern K, et al.: The identification of high-performing antibodies for Vacuolar protein sorting-associated protein 35 (hVPS35) for use in Western Blot, immunoprecipitation and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 452. Publisher Full Text

[19] 19. Ayoubi R, Alshafie W, You Z, et al.: The identification of high-performing antibodies for Superoxide dismutase [Cu-Zn] 1 (SOD1) for use in Western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 391. Publisher Full Text

[20] 20. Alshafie W, McPherson P, Laflamme C: Antibody screening by Immunofluorescence. Zenodo. 2021. Publisher Full Text

[21] 21. Fotouhi M, Alshafie W, Shlaifer I, et al.: Antibody Characterization Report for Charged multivesicular body protein 2b. [Dataset]. Zenodo. 2022. Publisher Full Text

[22] 22. Southern K: Dataset for the Charged multivesicular body protein 2b antibody screening study [Dataset]. Zenodo. 2023. Publisher Full Text

[23] 23. Bandrowski A, Pairish M, Eckmann P, et al.: The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2022; 51: D358–D367. Publisher Full Text

[24] 24. Bairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29(2): 25–38. PubMed Abstract | Publisher Full Text | Free Full Text

[25] 25. Ayoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot. Zenodo. 2021. Publisher Full Text

[26] 26. Ayoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation. Zenodo. 2021. Publisher Full Text

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

Abstract

Keywords

Introduction

Results and discussion

Table 1. Summary of the cell lines used.

Figure 1. Charged multivesicular body protein 2b antibody screening by Western Blot.

Figure 2. Charged multivesicular body protein 2b antibody screening by immunoprecipitation.

Figure 3. Charged multivesicular body protein 2b antibody screening by immunofluorescence.

Methods

Antibodies

Table 2. Summary of the Charged multivesicular body protein 2b antibodies tested.

CRISPR/Cas9 genome editing

Cell culture

Antibody screening by Western Blot

Antibody screening by immunoprecipitation

Antibody screening by immunofluorescence

Data availability

Underlying data

Acknowledgments

References

Comments on this article Comments (0)

Open Peer Review

Comments on this article Comments (0)

Open Peer Review

Reviewer Status

Reviewer Reports

Comments on this article

Browse by related subjects

Competing Interests Policy

Stay Updated