All lanes : Anti-N Cadherin antibody - Intercellular Junction Marker (ab18203) at 1 µg/ml

Lane 1 : Brain (Rat) Tissue Lysate at 10 µg
Lane 2 : Brain (Mouse) Tissue Lysate at 10 µg
Lane 3 : Brain (Human) Tissue Lysate at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 100 kDa
Observed band size: 125 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab18203 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

The N Cadherin protein has a predicted molecular weight of 100 kDa, however it is extensively glycosylated and has been shown to run in the 125-135 kDa region (SwissProt data).

Anti N-cadherin (ab18203) staining of mouse brain using immunohistochemistry (formaldehyde-fixed, paraffin-embedded sections). Heat-mediated antigen retrieval was carried out using citric acid. Samples were incubated with primary antibody (1/1000) for two hours at room temperature. A biotin-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

Anti N-cadherin (ab18203) staining of human ovarian cancer tissue using immunohistochemistry (formaldehyde-fixed, paraffin-embedded sections). Heat-mediated antigen retrieval was carried out using citric acid. Samples were incubated with primary antibody (1/1000) for two hours at room temperature. A biotin-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

Anti N-cadherin (ab18203) staining in a human melanoma xenograft mouse model using immunohistochemistry (formaldehyde-fixed, paraffin-embedded sections). Heat-mediated antigen retrieval was carried out using citric acid. Samples were incubated with primary antibody (1/1000) for two hours at room temperature. A biotin-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

Anti N-cadherin (ab18203) staining of E17 developing rat retina using immunohistochemistry (formaldehyde-fixed, paraffin-embedded sections). Heat-mediated antigen retrieval was carried out using citric acid. Samples were incubated with primary antibody (1/1000) for two hours at room temperature. A biotin-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

IHC image of N Cadherin staining in Human liver cancer formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab18203, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Anti-N Cadherin antibody - Intercellular Junction Marker (ab18203) stained human embryonic stem cells differentiated into mesoderm.

N Cadherin was immunoprecipitated using 0.5mg Mouse Brain whole tissue lysate, 5µg of Rabbit polyclonal to N Cadherin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse Brain whole tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab18203.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 135kDa: N Cadherin

ab18203 staining N Cadherin in Human liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 10% serum for 30 minutes at 25°C; antigen retrieval was by heat mediation in a 10 mM citrate buffer pH6.0. Samples were incubated with primary antibody (1/100 in PBS plus casein) for 90 minutes at 37°C. A Biotin-conjugated Goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.

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Paraformaldehyde-fixed, 0.2% Triton X100 permeabilized HaCaT (human keratinocyte cell line) cells stained for N Cadherin (green) using ab18203 at 1/200 dilution in ICC/IF, followed by Donkey anti Rabbit Alexa Fluor 568 at 1/500 dilution.

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Immunohistochemistry of kidney carcinoma staining N Cadherin with ab18203 at 1μg/ml.