Recombinant Anti-Metabotropic Glutamate Receptor 5 antibody [EPR2425Y] (ab76316)

IHC image of Metabotropic Glutamate Receptor 5 staining in a section of frozen normal human hippocampus* performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab76316, 1 µg/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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

All lanes : Anti-Metabotropic Glutamate Receptor 5 antibody [EPR2425Y] (ab76316) at 1/10000 dilution

Lane 1 : Human spinal cord tissue lysate was not boiled before loading to the gel
Lane 2 : Human hippocampus tissue lysate was not boiled before loading to the gel
Lane 3 : Human liver tissue lysate was not boiled before loading to the gel
Lane 4 : Human spinal cord tissue lysate was boiled before loading to the gel
Lane 5 : Human hippocampus tissue lysate was boiled before loading to the gel
Lane 6 : Human liver tissue lysate was boiled before loading to the gel

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 132 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?
Additional bands at: 280 kDa (possible dimer)

Buffer: 5% NFDM /TBST

Please do not boil the sample before loading to the gel. 

GRM5 is a brain enriched protein, can be observed in most of the brain areas but low in spinal cord (PMID: 12783878).

Anti-Metabotropic Glutamate Receptor 5 antibody [EPR2425Y] (ab76316) at 1/10000 dilution + mouse brain lysate at 10 µg

Secondary
Goat anti-rabbit-HRP

Developed using the ECL technique.

Predicted band size: 132 kDa
Observed band size: 140-150 kDa why is the actual band size different from the predicted?
Additional bands at: >250 kDa (possible dimer)

 

Immunohistochemical analysis of Paraffin-embedded rat cerebrum tissue sections labeling Metabotropic Glutamate Receptor 5 with ab76316 at 1/400 dilution followed by Goat Anti-Rabbit IgG H&L (HRP Polymer) secondary antibody. Sections were counterstained with Hematoxylin. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0).

Positive staining on rat cerebrum.

ab76316 staining Metabotropic Glutmate Receptor 5 in rat caudate putamen by immunohistochemistry. Tissue was fixed with formaldehyde and citrate-mediated antigen retrieval was performed. Samples were blocked with 1% BSA for 10 minutes at 21°C, before incubation with the primary antibody (1/2000) for 2 hours at 21°C. A biotin conjugated goatanti-rabbit IgG secondary was used at 1/250.

See Abreview

ab76316 staining Metabotropic Glutmate Receptor 5 in mouse caudate putamen/ Corpus callosum by immunohistochemistry (frozen sections). Tissue was fixed with formaldehyde and samples were blocked with 1% BSA for 10 minutes at 21°C, before incubation with the primary antibody (1/2000) for 16 hours at 21°C. An alexa fluor® 594 conjugated goat anti-rabbit IgG secondary was used at 1/500.

See Abreview

Postembedding immunogold labeling of mouse neocortex using Anti-Metabotropic Glutamate Receptor 5 antibody [EPR2425Y] (ab76316). The tissue was embedded in Lowicryl HM20 resin. 60 nm sections were then cut and mounted on nickel mesh grids before undergoing antigen retrieval for 15 minutes in 0.01 M citrate buffer, pH 6 at 60°C.

The sections were then blocked in 1% BSA/TBSN pH 7.6 and incubated overnight at room temperature with Anti-Metabotropic Glutamate Receptor 5 antibody [EPR2425Y] (ab76316) at 1:250. Sections were washed twice in TBSN pH 7.6, treated with 1% normal donkey serum/TBSN pH 8.2 for 30 minutes, before being incubated with donkey anti-rabbit IgG-Au 10-20 nm at 1:20 for two hours at room temperature.

Sections were then washed in TBSN pH 8.2, followed by water, before undergoing post-staining with 1% uranyl acetate and Sato’s lead. They were then air dried before being transferred to an oven for 30 minutes at 60°C.

In these images you can see gold immunoparticles on the postsynaptic density of synapses.

(TBSN = 0.02M TRIS buffered saline (0.3 N, pH 7.6 or 8.2) with 0.005% Tergitol NP-10)

Intracellular flow cytometric analysis of permeabilized SH-SY5Y cells using ab76316 (red) at 1/20 or a rabbit IgG (ab172730) as a negative control (green). The cells were permeabilized with 2% PFA and a goat anti-rabbit IgG FITC was used as the secondary at 1/150. 

 

 

ab76316 staining Metabotropic Glutmate Receptor 5 in mouse caudate putamen/ Corpus callosum by immunohistochemistry. Tissue was fixed with formaldehyde and citrate-mediated antigen retrieval was performed. Samples were blocked with 1% BSA for 10 minutes at 21°C, before incubation with the primary antibody (1/1000) for 2 hours at 21°C. A biotin conjugated goatanti-rabbit IgG secondary was used at 1/250.

See Abreview