This is a knockout-validated antibody summary, based on two publications "Tissue-specific cross-reactivity of connexin32 antibodies: problems and solutions unique to the central nervous system" [1] and "Gap junction mediated intercellular metabolite transfer in the cochlea is compromised in connexin30 null mice" [2]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

Rabbit polyclonal

Company: Zymed

Antibody: Cx30

Catalog number: 71-2200

Summary: Rabbit polyclonal against a synthetic peptide derived from the unique C-terminus of the mouse Connexin 30. Reacts with human, mouse, rat, cat, chicken, and guinea pig. Suitable for western blot, immunofluorescence, immunohistochemistry, and ELISA.

Western blot

Brain and liver tissues from human and WT, and Cx30 knockout mice. All tissues were homogenized in fresh RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM NaF, 50 g/ml aprotinin, 1 mM sodium orthovanadate, 1 mg/ml phenylmethylsulfonyl fluoride, 10 mM phosphate-buffered saline (PBS; 10 mM phosphate, 154 mM NaCl)).

5% (w/v) skim milk powder–PBS with 0.1% Tween-20 (PBST; blocking buffer) for 1 to 3 h.

1:200 dilution in blocking buffer overnight at 4°C.

Horseradish peroxidase (HRP)-conjugated anti-mouse (Jackson ImmunoResearch

Laboratories, PA; 1:2,000) diluted in blocking buffer for 1- to 3-h.

X-ray film using SuperSignal West Pico Chemiluminescent Substrate (Pierce, IL).

Please see Figure 8b in the article [1].

Immunohistochemistry

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Figure 1. (A) Immunoreactivity of Cx26 showing the remaining GJ plaques in the Claudius cells (one example is given by a small white arrow) and outer sulcus cells (white arrowhead) in the cochlea of the Cx30 null mice. (B) Immunoreactivity of Cx30 obtained from the same view as that in (A). Focus was not changed. The antibody against Cx30 was confirmed to generate positive results in the WT animals. (C) Differential interference contrast (DIC) image of the same cochlear tissue as that obtained in (A). Focus was not changed. Regions of the outer sulcus cells and Claudius cells are labeled. (D) Fluorescent image of DAPI showing the nuclei of the cochlear cells. The image was obtained from the same cochlear tissue as that in (A) with the same focus. Borders of the region of outer sulcus cells and Claudius cells are outlined by white lines and labeled. From [2]. Note that positive control is not shown in the image, but mentioned in the caption.

Cochlea of Cx30 null mice. Tissue was flattened and fixed in 4% paraformaldehyde.

10% goat serum (in PBS) for 1 hour.

1∶100 dilution overnight at 4°C.

FITC-conjugated (catalog# 111-096-003) goat anti rabbit secondary antibodies (Jackson ImmunoResearch Lab, West Grove, PA) for 2 hours at room temperature.

Conventional fluorescent microscope.

If the antibody described in this summary is a polyclonal antibody, since polyclonal antibodies are of limited quantity, please inquire the supplier whether any current polyclonal antibody with the same catalog number is exactly the same as the one described in this summary. Sometimes, different bleeds or different animals are used, usually with a different lot number. In such cases, the result in this summary may not apply to the new antibody with the same catalog number.