Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72

Abstract

Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties.

C9orf72-associated SMCR8 protein binds in the ubiquitin pathway and with proteins linked with neurological disease

“…S1 B-E), plus additional bands of unknown specificicy, but which could in part relate to the above desribed SMCR8 protein isoforms. The Bethyl and Abcam ab202283 α-SMCR8 antibodies have been used in other studies, and our observations are similar [ 34 , 50 , 65 , 79 ]. Interestingly, although expression of full length SMCR8 protein was detected in human brain tissues, none was seen in spinal cord tissue lysates of multiple samples (Fig.…”

“…S4 F.G). To confirm further these observations, two polyclonal antibodies developed by the Robertson lab [ 19 , 79 ], and specific for C9-L (Fig. 4 f) and C9-S (not shown) isoforms, were also tested but failed to show obvious C9orf72 protein presence in TIA1-marked SGs in DTT- or NaAsO 2 -stressed cells of multiple lines, including U2OS and 2012Ep cells.…”

“…S1 A). A small number of non-commercial C9orf72-specific antibodies have also been described [ 17 , 23 , 30 , 79 ].…”

“…Commercial antibodies included mouse (ms) α-V5-tag (Invitrogen), rabbit (rb) α-DYKDDDDK-tag (α-FLAG) (D6W5B), ms α-HA-tag (6E2), rb α-Myc-tag (71D10), rb α-HSP90, and ms α-Ubiquitin (UBB) (P4D1) (Cell Signaling Technology), rb α-4E-T, rb α-C9orf72 (S-14), goat (gt) α-eIF3η (N-20), ms α-p70 S6 kinase (which recognizes HEDLS/EDC4 [77]), gt α-TIA1 (C-20), and rb α-WDR41 (S-12) (Santa Cruz Biotechnology), rb α-SMCR8 (ab186504, epitope region 841-890) and rb α-SMCR8 (ab202283, epitope region 600-650) (Abcam), rb α-C9orf72 (22637-1-AP), rb α-SMCR8 (21125-1-AP, epitope region 588-937), and rb α-WDR41 (26817-1-AP) (Proteintech), and rb α-SMCR8 (A304-694A, epitope region 600-650) (Bethyl). Monoclonal ms α-4H1-ORF1 was kindly provided by Dr. K. Burns (JHU, USA, also Millipore MABC1152, [78]), rb α-C9-L and rb α-C9-S antibodies by Dr. J. Robertson (U. Toronto, Canada, [19,79]), and α-GW182 human serum (IC-6) by Dr. M. Fritzler (U. Calgary, [80]). Donkey Cy3-, DyLight 488-, or DyLight 549-conjugated, and HRP-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories.…”

Repeat length increases disease penetrance and severity in C9orf72 ALS/FTD BAC transgenic mice

“…Commercially available antibodies detect the long isoform. We used the C9orf72 Genetex antibody that detects both human and mouse C9orf72 proteins (Laflamme, McKeever et al, 2019) and confirmed antibody specificity by showing that the 55kDa protein is detected in the control but not C9orf72 KO brain lysates (generously donated by Dr. Robert. H. Baloh) ( Fig 1C).…”