Anti-Metabotropic Glutamate Receptor 3/MGLUR3 antibody (ab140741)

IHC image of Metabotropic Glutamate Receptor 3/MGLUR3 staining in mouse brain formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol B. 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 ab140741, 5µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC 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-Metabotropic Glutamate Receptor 3/MGLUR3 antibody (ab140741) at 1 µg/ml

Lane 1 : Brain (Mouse) Tissue Lysate
Lane 2 : Brain (Rat) Tissue Lysate
Lane 3 : Human brain tissue lysate - total protein (ab29466)
Lane 4 : Mouse Hippocampus Tissue Lysate
Lane 5 : Rat Hippocampus Tissue Lysate
Lane 6 : Mouse Cerebellum Tissue Lysate
Lane 7 : Rat Cerebellum Neuronal cell lysate

Lysates/proteins at 10 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: 99 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

The band observed at 95 kDa could potentially be a cleaved form of Metabotropic Glutamate Receptor 3/MGLUR3 due to the presence of a 22 amino acid signal peptide.

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 5% Bovine Serum Albumin before being incubated with ab140741 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.