Home Primary Antibodies Metabotropic Glutamate Receptor 2 + 3 Antibody [K11L17]

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Metabotropic Glutamate Receptor 2 + 3 Antibody [K11L17]

Catalog No.: F3975

    Application: Reactivity:
    • F3975-wb
      Lane 1: Mouse brain

    Experiment Essentials

    WB
    Recommended SDS-PAGE separating gel concentration: 5%.
    Recommended wet transfer conditions: 250 mA, 180 min.
    Recommended WB dilution ratio: 1:10000

    Usage Information

    Dilution
    1:10000 - 1:50000
    1:1800
    1:1000
    1:70
    Application
    WB, IHC, IF, FCM
    Reactivity
    Mouse, Rat, Human
    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
    Observed MW
    95 kDa, 250 kDa
    *Why do the predicted and actual molecular weights differ?
    The following reasons may explain differences between the predicted and actual protein molecular weight.
    Positive Control Mouse primary neurone cell; Human cerebrum tissue; Mouse cerebrum tissue; Rat cerebrum tissue; Human brain (unboiled); Mouse brain (unboiled); Rat brain (unboiled)
    Negative Control Rat brain (boiled); Mouse brain (boiled)

    Exprimental 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.
    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) and put the sample on ice for 5 min.
    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) and put the sample on ice for 5 min.
    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. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.
     
    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: 250 mA, 180 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 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:10000), 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.
    IF
    Experimental Protocol：
     
    Specimen Preparation 
    1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS.
    NOTE: Paraformaldehyde is toxic, use only in a fume hood.
    2. Fix cells for 15 min at room temperature.
    3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
    4. Proceed with Immunostaining.
     
    Immunostaining
    1. Add theblocking buffer and incubate for 60 min at RT.
    2. Prepare primary antibody diluent in antibody dilution buffer as recommended .
    3. Aspirate blocking solution, apply diluted primary antibody.
    4. Incubate overnight at 4°C.
    5. Rinse three times in 1X PBS for 5 min each.
    6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
    7. Rinse three times in 1X PBS for 5 min each.
    8. Mount slides usingmounting medium with DAPI and cover with coverslips.
    9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 23°C protected from light.
     
    IHC
    Experimental Protocol：
     
    Deparaffinization/Rehydration
    1. Deparaffinize/hydrate sections:
    2. Incubate sections in three washes of xylene for 5 min each.
    3. Incubate sections in two washes of 100% ethanol for 10 min each.
    4. Incubate sections in two washes of 95% ethanol for 10 min each.
    5. Wash sections two times in dH2O for 5 min each.
    6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
     
    Staining
    1. Wash sections in dH2O three times for 5 min each.
    2. Incubate sections in 3% hydrogen peroxide for 10 min.
    3. Wash sections in dH2O two times for 5 min each.
    4. Wash sections in wash buffer for 5 min.
    5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
    6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
    7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
    8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
    9. Wash sections three times with wash buffer for 5 min each.
    10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
    11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
    12. Immerse slides in dH2O.
    13. If desired, counterstain sections with hematoxylin.
    14. Wash sections in dH2O two times for 5 min each.
    15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.
    16. Mount sections with coverslips and mounting medium.
     

    Datasheet & SDS

    Biological Description

    Specificity
    Metabotropic Glutamate Receptor 2 + 3 Antibody [K11L17] detects endogenous levels of total Metabotropic Glutamate Receptor 2 and 3 protein.
    Subcellular Location
    Cell membrane, Cell projection, Membrane, Synapse
    Uniprot ID
    Q14416
    Clone
    K11L17
    Synonym(s)
    GPRC1B; MGLUR2; GRM2; Metabotropic glutamate receptor 2; mGluR2
    Background
    Metabotropic Glutamate Receptors 2 and 3 (mGlu2/3) are group II metabotropic glutamate receptors and members of the class C G protein-coupled receptor (GPCR) family, widely expressed in the central nervous system (CNS), where they modulate excitatory synaptic transmission and neuronal excitability. Each receptor forms a homodimer composed of two subunits, each containing a large extracellular Venus flytrap (VFT) ligand-binding domain, a cysteine-rich domain (CRD), and a seven-transmembrane domain (TMD) responsible for intracellular signaling. Although mGlu2 and mGlu3 share high sequence homology, they differ subtly in ligand affinity and regulatory mechanisms: mGlu3 can detect lower glutamate concentrations and is uniquely regulated by specific phosphorylation and dephosphorylation events. They primarily couple to Gi/o proteins, leading to inhibition of adenylyl cyclase, reduction of cAMP production, and downstream suppression of protein kinase A activity. They also activate MAPK/ERK pathways, potentially via Gβγ subunits, and regulate ion channels by inhibiting voltage-gated Ca2+ channels while activating K+ channels. As presynaptic autoreceptors, mGlu2/3 dynamically inhibit glutamate release to maintain synaptic homeostasis. They also act as heteroreceptors, modulating the release of various neurotransmitters, including monoamines and neuropeptides, and contribute to synaptic plasticity phenomena such as long-term depression (LTD). mGlu2 and mGlu3 receptors exist in distinct conformational states stabilized by agonists and antagonists. Ligand binding triggers conformational changes that are transmitted from the Venus flytrap domain (VFT) to the transmembrane domain (TMD), activating the receptor through an anticlockwise rotation of the transmembrane dimer. In the case of mGlu2 and mGlu3 antagonism, negative allosteric modulators induce alternate TMD conformations that represent inactive states distinct from those of other mGlu receptors. Dysregulation of mGlu2/3 signaling is implicated in various neuropsychiatric disorders.
    References
    • https://pubmed.ncbi.nlm.nih.gov/35236939/
    • https://pubmed.ncbi.nlm.nih.gov/36407098/

    Tech Support

    Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

    Handling Instructions

    Tel: +1-832-582-8158 Ext:3
    If you have any other enquiries, please leave a message.

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