This is a knockout-validated antibody summary, based on the publication "Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

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Figure 1. Cell lysates from HEK-293 parental, heterozygous (+/-) or two individual C9ORF72 KO clones (KOA, KOB) were prepared and processed for immunoblot with the indicated C9ORF72 antibodies. The arrows point to positive C9ORF72 signals. The Ponceau stained transfers associated with each blot are shown as a protein loading control. lysis of 16 C9ORF72 antibodies by immunoblot.

Mouse monoclonal IgG1

Company: GeneTex

Antibody: C9orf72

Catalog number: GTX632041

Summary: Mouse monoclonal IgG1 against human C9orf72. Reacts with mouse, rat and human. Suitable for western blot, immunoprecipitation, immunocytochemistry/immunofluorescence and immunohistochemistry (paraffin).

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Figure 2. Longer exposure immunoblots on HEK-293 cell lysates and immunoblots of brain lysates from WT and C9ORF72 KO mice with the indicated C9ORF72 antibodies. The arrows point to positive C9ORF72 signals.

Western blot | Immunocytochemistry | Immunohistochemistry

HEK-293 parental, heterozygous (+/-) and C9ORF72 KO clones, and WT and C9ORF72 KO brain tissues.

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Figure 3. Parental and KO cells were transfected with LAMP1-YFP or LAMP1-RFP, respectively. Parental and KO cells were combined and incubated with buffer only or stained with the indicated GeneTex C9ORF72 monoclonal antibodies. Grayscale images of the green, red and far-red channels are shown. Parental and KO cells are outlined with green and red dashed line, respectively. Representative images are shown. Bars = 40 um.

WB: Cultured cells were collected in HEPES lysis buffer (20 mM HEPES, 100 mM sodium chloride, 1 mM EDTA, 5% glycerol, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor.

ICC: Cells were fixed in 4% PFA for 10 min.

IHC: Formalin-fixed, paraffin-embedded brain tissue from 3 month old C9-KO (n = 3) and C9-WT (n = 3) mice were sectioned, deparaffinized and heat-induced epitope retrieval was performed.

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Figure 4. DAB immunohistochemistry of C9ORF72 WT and C9ORF72 KO mouse brains in the sagittal plane of the indicated brain regions using C9ORF72 antibody GTX632041. Scale bars = 100 um. From [1].

WB: 5% milk in TBS with 0.1% Tween 20 (TBST).

ICC: TBS, 5% BSA and 0.3% Triton X-100, pH 7.4 for 1 hr at room temperature.

IHC: 2.5% normal horse serum and 0.3% Triton X-100 in TBS for 1 hr at room temperature.

WB: 5% bovine serum albumin in TBS with 0.1% Tween 20 (TBST) O/N at 4˚C.

ICC: blocking buffer containing the primary C9ORF72 antibodies diluted at 2 µg/ml and incubated O/N at 4°C.

IHC: diluted in DAKO antibody diluent (Agilent, Cat# S0809) at 1:5,000 and incubated O/N at 4°C.

WB: 1:10,000 dilution anti-rabbit (Jackson ImmunoResearch Laboratories) in TBST with 5% milk for 1 hr at room temperature.

ICC: Alexa Fluor 647-conjugated secondary antibodies diluted 1:1,000 in blocking buffer for 2 hr at room temperature.

IHC: ImmPRESS HRP horse anti-mouse IgG (Vector Labs, Cat# MP-7402), for 1 hr at room temperature.

WB: Detection of immuno-reactive bands was performed by image scan using a LI-COR Odyssey Imaging System (LI-COR Biosciences). Please note there are minor unspecific bands in WB.

ICC: Imaging was performed using a Leica SP8 laser scanning confocal microscope equipped with a 40x oil objective (NA = 1.30) and HyD detectors.

IHC: ImmPACT DAB peroxidase substrate kit (Vector Labs, Cat# SK-4105). Slides were then counterstained with Hematoxylin Solution, Gill No.1 (Sigma-Aldrich, Cat# GHS132) for 5 min at room temperature. Micrographs were captured with a digital camera mounted on a Leica DM6000 B upright microscope with either 10X or 40X objectives using Volocity Imaging software (version 6.3.0 PerkinElmer).