Home Primary Antibodies Anti-EAAT2 Rabbit Antibody [C5P1] For research use only.

Anti-EAAT2 Rabbit Antibody [C5P1]

Catalog No.: F3745

    Application: Reactivity:

    Usage Information

    Dilution
    1:1000
    1:30
    1:2000
    Application
    WB, IP, IHC
    Reactivity
    Mouse, Rat
    Source
    Rabbit
    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
    62 kDa 180 kDa,65 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 brain; Rat brain; Mouse striatum tissue; Mouse kidney tissue; Rat striatum tissue
    Negative Control Mouse liver; Mouse kidney; Rat liver; Neuro-2a; C6; RAW 264.7; PC-12; NIH/3T3

    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: 10%. 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 5% skim milk powder 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.
    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
    EAAT2 Rabbit mAb detects endogenous levels of total EAAT2 protein.
    Subcellular Location
    Cell membrane, Membrane
    Uniprot ID
    P43004
    Clone
    C5P1
    Synonym(s)
    Eaat2, Glt1, Slc1a2, Excitatory amino acid transporter 2, GLT-1, Sodium-dependent glutamate/aspartate transporter 2, Solute carrier family 1 member 2
    Background
    EAAT2 (Excitatory Amino Acid Transporter 2), also known as SLC1A2, is a major glutamate transporter in the central nervous system and a key member of the solute carrier family 1 (SLC1). It is primarily expressed in astrocytes and is responsible for clearing of extracellular glutamate, thereby maintaining synaptic glutamate homeostasis. EAAT2 functions as a trimer, with each protomer composed of a central scaffold domain involved in trimerization and membrane anchoring, and a transport domain that contains eight transmembrane helices and two helical hairpin loops, HP1 and HP2. The glutamate-binding site is situated between HP1 and HP2, and six conserved residues, Ser364, Thr401, Asp475, Arg478, Thr479, and Asn482, are essential for high-affinity glutamate binding. EAAT2 operates through an “elevator-like” alternating access mechanism: upon glutamate binding and closure of the HP2 loop, the transport domain shifts to move the substrate into the cell. This transport is tightly coupled with the co-transport of three Na⁺ ions and one H⁺ ion into the cell, and the counter-transport of one K⁺ ion out, resulting in electrogenic glutamate uptake. EAAT2 is enriched at the astrocyte plasma membrane, especially in cholesterol-rich lipid raft microdomains, where cholesterol enhances its activity by facilitating the conformational dynamics required for transport. By efficiently removing excess glutamate, EAAT2 protects neurons from excitotoxicity, a process that contributes to the pathology of several neurological disorders such as amyotrophic lateral sclerosis (ALS), epilepsy, cerebral ischemia, and other neurodegenerative diseases. Reduced expression or dysfunction of EAAT2 impairs glutamate clearance, leading to elevated extracellular glutamate levels and increased neuronal vulnerability.
    References
    • https://pubmed.ncbi.nlm.nih.gov/26033496/
    • https://pubmed.ncbi.nlm.nih.gov/35953475/

    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
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