IHC image of Junctional Adhesion Molecule 2/JAM-B staining in Human normal heartmuscle formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab139645, 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.

All lanes : Anti-Junctional Adhesion Molecule 2/JAM-B antibody (ab139645) at 1 µg/ml (Milk blocking - 3%)

Lane 1 : Heart (Human) Tissue Lysate - adult normal tissue
Lane 2 : Heart (Mouse) Tissue Lysate
Lane 3 : Heart (Rat) Tissue Lysate

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?


Exposure time: 8 minutes

Junctional Adhesion Molecule 2/JAM-B contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

The predicted molecular weight of Junctional Adhesion Molecule 2/JAM-B is 33 kDa (SwissProt), however we expect to observe a banding pattern around 45 kDa.

This blot was produced using a 10% 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 3% Milk before being incubated with ab139645 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.