mGluR5 Antibody [D5P24] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat

Lane 1: Mouse brain
Immunofluorescent analysis of Mouse hippocampus tissue using F8612 (green, 1:200), Hoechst (blue) and tubulin (Red).

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Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.
Exposure time of at least 90s is recommended.

Usage Information

Dilution
WB1:1000 IP1:50 IF1:200

Application
WB, IP, IF

Reactivity
Human, Mouse, Rat

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
132 kDa 300 kDa, 150 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Mouse hippocampus; Mouse brain; Huamn cancer; Human cerebellum; Human iPSC-derived cortical glutamatergic neurons
Negative Control	Mouse lung; Mouse striatum

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system. (Exposure time of at least 90s is recommended)

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system. (Exposure time of at least 90s is recommended)

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

IF

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

Datasheet & SDS

Biological Description

Specificity
mGluR5 Antibody (Rabbit mAb) [D5P24] detects endogenous levels of total mGluR5 protein.

Subcellular Location
Cell membrane, Membrane

Uniprot ID
P41594

Clone
D5P24

Synonym(s)
glutamate metabotropic receptor 5 \| glutamate receptor \| metabotropic 5 \| GPRC1E \| GRM5 \| Metabotropic glutamate receptor 5 \| mGlu5 \| MGLUR5 \| PPP1R86 \| protein phosphatase 1 \| regulatory subunit 86

Background
Metabotropic glutamate receptor 5 (mGluR5) is a group I class C G protein‑coupled glutamate receptor that belongs to the GRM1/GRM5 subfamily and couples primarily to Gq/11 to regulate excitatory synaptic transmission and plasticity in the central nervous system. The receptor contains a large bilobed extracellular Venus flytrap domain that binds glutamate, a cysteine‑rich linker, a seven‑transmembrane helical core, and an intracellular C‑terminal tail that associates with scaffold and signaling proteins at postsynaptic densities and at intracellular membranes. Activation by glutamate induces conformational changes that promote Gq/11‑dependent stimulation of phospholipase C, generation of inositol 1,4,5‑trisphosphate and diacylglycerol, release of Ca²⁺ from intracellular stores, and activation of protein kinase C, with additional engagement of mitogen‑activated protein kinase cascades and protein kinase D phosphorylation that extends the temporal range of mGluR5 signaling. The receptor localizes at perisynaptic regions of excitatory synapses where it interacts functionally with NMDA receptor signaling to modulate synaptic strength, dendritic excitability, and long‑term forms of synaptic plasticity, and intracellular pools of mGluR5 on endomembranes and nuclear compartments couple to distinct Gq/11‑linked pathways that generate localized Ca²⁺ signals and transcriptional responses associated with sustained synaptic activity. mGluR5 is expressed at high levels in cortical, hippocampal, and striatal neurons and is also detectable in astrocytes and microglia, where receptor activation influences glutamate handling, neuroglial signaling, and the balance between pro‑ and anti‑inflammatory outputs, placing this receptor at the interface of neuronal and glial regulation of circuit excitability. The C‑terminal domain forms complexes with PSD‑95, Homer, and SHANK family proteins that organize receptor positioning in perisynaptic nanodomains and couple mGluR5 activation to downstream effectors controlling spine morphology, receptor trafficking, and gene expression programs linked to learning and memory and to stress‑related behavioral responses. Dysregulated mGluR5 signaling associates with fragile X‑related synaptic dysfunction, addiction, anxiety, mood disorders, neurodegeneration, and neuropathic pain, where altered receptor expression or signaling bias contributes to changes in synaptic plasticity, nociceptive processing, or emotional behavior.

Background

Metabotropic glutamate receptor 5 (mGluR5) is a group I class C G protein‑coupled glutamate receptor that belongs to the GRM1/GRM5 subfamily and couples primarily to Gq/11 to regulate excitatory synaptic transmission and plasticity in the central nervous system. The receptor contains a large bilobed extracellular Venus flytrap domain that binds glutamate, a cysteine‑rich linker, a seven‑transmembrane helical core, and an intracellular C‑terminal tail that associates with scaffold and signaling proteins at postsynaptic densities and at intracellular membranes. Activation by glutamate induces conformational changes that promote Gq/11‑dependent stimulation of phospholipase C, generation of inositol 1,4,5‑trisphosphate and diacylglycerol, release of Ca²⁺ from intracellular stores, and activation of protein kinase C, with additional engagement of mitogen‑activated protein kinase cascades and protein kinase D phosphorylation that extends the temporal range of mGluR5 signaling. The receptor localizes at perisynaptic regions of excitatory synapses where it interacts functionally with NMDA receptor signaling to modulate synaptic strength, dendritic excitability, and long‑term forms of synaptic plasticity, and intracellular pools of mGluR5 on endomembranes and nuclear compartments couple to distinct Gq/11‑linked pathways that generate localized Ca²⁺ signals and transcriptional responses associated with sustained synaptic activity. mGluR5 is expressed at high levels in cortical, hippocampal, and striatal neurons and is also detectable in astrocytes and microglia, where receptor activation influences glutamate handling, neuroglial signaling, and the balance between pro‑ and anti‑inflammatory outputs, placing this receptor at the interface of neuronal and glial regulation of circuit excitability. The C‑terminal domain forms complexes with PSD‑95, Homer, and SHANK family proteins that organize receptor positioning in perisynaptic nanodomains and couple mGluR5 activation to downstream effectors controlling spine morphology, receptor trafficking, and gene expression programs linked to learning and memory and to stress‑related behavioral responses. Dysregulated mGluR5 signaling associates with fragile X‑related synaptic dysfunction, addiction, anxiety, mood disorders, neurodegeneration, and neuropathic pain, where altered receptor expression or signaling bias contributes to changes in synaptic plasticity, nociceptive processing, or emotional behavior.

References
https://pubmed.ncbi.nlm.nih.gov/11880516/ https://pubmed.ncbi.nlm.nih.gov/23205012/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%

mGluR5 Antibody (Rabbit mAb) [D5P24]