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Immunoprecipitation/ IP Kit - Anti-EphB3 / HEK2 / Eph Receptor B3 Immunomagnetic Beads

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    Immunoprecipitation/ IP Kit- Anti-EphB3 / HEK2 / Eph Receptor B3 Immunomagnetic Beads
    Product Contents
    ContentsPackage 1Package 2Storage
    Anti-EphB3/HEK2/Eph Receptor B3 Immunomagnetic Beads1 31 mL5 mL2-8℃ for 12 months
    NP40 Cell Lysis Buffer4 mL22 mL-20℃ for 12 months
    5×TBST(pH7.4)Required but not supplied
    1×TBST(pH7.4)Required but not supplied
    ddH2ORequired but not supplied
    EphB3/HEK2/Eph Receptor B3 Positive Cell Lysis300 μg300 μg-20℃ for 12 months
    Alkaline Elution Buffer3 mL15 mL2-8℃ for 12 months
    Acidity Elution Buffer3 mL15 mL2-8℃ for 12 months
    Neutralization Buffer2 mL8 mL2-8℃ for 12 months
    Magnetic Separator Not included (refer related product MAGS001)One MAGS001 included in China2

    [1] The IP KIT contains anti-EphB3/HEK2/Eph Receptor B3 Immunomagnetic Beads(2 mg/mL) in phosphate buffered saline (PBS, pH 7.4) with sodium azide (0.1%).
    [2] The Magnetic Separator cannot be included for oversea customers due to shipment prohibition.

    [3] Shipping: Immunomagnetic Beads kits are shipped at ambient temperature in which immunomagnetic beads are provided in liquid buffer.

    Product Description

    The Anti-EphB3/HEK2/Eph Receptor B3 Immunomagnetic Beads, conjugated with Anti-EphB3/HEK2/Eph Receptor B3 antibody, are used for immuneprecipitation (IP) of EphB3/HEK2/Eph Receptor B3 proteins which expressed in vitro expression systems and bacterial and mammalian cell lysates. For IP, the beads are added to a sample containing EphB3/HEK2/Eph Receptor B3 proteins to form a bead-protein complex. The complex is removed from the solution manually using a Magnetic Separator. The bound EphB3/HEK2/Eph Receptor B3 proteins are dissociated from the Immunomagnetic Beads using an elution buffer.

    Antibody Information
    Antibody:81593-T36
    Immunogen:Recombinant Rat EphB3 / HEK2 / Eph Receptor B3 Protein (Catalog#81593-R08H)
    Isotype:Rabbit IgG
    分子種屬:Rat
    产品或服务:1
    Clone ID:
    Molecule:EphB3
    抗體製備方法:Produced in rabbits immunized with purified, recombinant Rat EphB3 / HEK2 / Eph Receptor B3 (rR EphB3 / HEK2 / Eph Receptor B3; Catalog#81593-R08H; NP_001099338.1; Met1-Thr537). EphB3 / HEK2 / Eph Receptor B3 specific IgG was purified by Rat EphB3 / HEK2 / Eph Receptor B3 affinity chromatography.
    特異性:Rat EphB3 / HEK2 / Eph Receptor B3
    Guaranteed Applications:IP,Minimum Protein Purification
    IP Protocol

    Fig. 1 Immunoprecipitation (IP) Protocol

    The protocol (Fig. 1) uses 50 μL Anti-EphB3/HEK2/Eph Receptor B3 Immunomagnetic Beads, but this can be scaled up or down as required.
    Cell Lysis
    Cells may be lysed using any standard cell lysis protocol in accordance with your starting materials. We suggest using NP40 Cell Lysis Buffer (supplied with kit).
    Immunoprecipitate Target Antigen
    1. Add 50 μL of Immunomagnetic Beads into a 1.5 mL microcentrifuge tube.
    2. Add 150 μL of 1× TBST buffer to the Immunomagnetic Beads and gently vortex to mix.
    3. Place the tube into a Magnetic Separator to collect the beads against the wall side of the tube. Remove and discard the supernatant.
    4. Add 1 mL of 1×TBST buffer to the tube. Invert the tube several times or gently vortex to mix for 1 min. Collect Immunomagnetic Beads with a Magnetic Separator. Remove and discard the supernatant.
    5. Add the sample containing target protein (100 μg of protein in 100 μL) to the pre-washed Immunomagnetic Beads, add 400 μL of 1×TBST buffer and incubate at room temperature for 30 min with mixing.
    6. Collect the Immunomagnetic Beads with a Magnetic Separator, remove the unbounded sample and save for analysis.
    7. Add 300 μL of 5× TBST buffer to the tube and gently mix. Collect the Immunomagnetic Beads and discard the supernatant. Repeat this wash twice. 8. Add 300 μL of ddH2O to the tube and gently mix. Collect the Immunomagnetic Beads on a Magnetic Separator and discard the supernatant.
    Elute Target Antigen.
    A. Alkaline Elution Protocols
    1. Add 100 μL of Alkaline Elution buffer to the tube.
    2. Gently vortex to mix and incubate the sample at room temperature on a rotator for 5 min.
    3. Magnetically separate the Immunomagnetic Beads and save the supernatant containing the target antigen.
    4. To neutralize the sample, add 50 μL of Neutralization Buffer for each 100 μL of eluate.
    B. Acidity Elution
    1. Add 100 μL Acidity Elution Buffer.
    2. Gently vortex to mix and incubate the sample at room temperature on a rotator for 5-10 min.
    3. Magnetically separate the Immunomagnetic Beads and save the supernatant containing the target antigen.
    4. To neutralize the low pH, add 15 μL of Neutralization Buffer for each 100 μL of eluate.
    C. Elution Using Sample Buffer
    1. Add 100 μL of SDS-PAGE sample buffer to the tube.
    2. Gently vortex to mix and incubate the sample at 95-100⁰C for 5-10 min.
    3. Magnetically separate the Immunomagnetic Beads and save the supernatant containing the antigen.

    Reference Information

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    Trouble Shooting

    ProblemPossible CauseSolution
    Little or no protein is detectedProtein degradedInclude protease inhibitors (PMSF) in the lysis buffer
    Use new lysate or lysate stored at -80℃
    No or minimal protein was expressedVerify protein expression by SDS-PAGE or Western blot
    analysis of the lysate using an positive control as a reference
    Increase the amount of lysate used for IP/Co-IP
    Use a more sensitive detection system
    Immunomagnetic Beads aggregatedImmunomagnetic beads were frozen or centrifugedHandle the Immunomagnetic Beads as directed in the instructions
    Buffer was incompatible with Immunomagnetic Beads
    Detergent was not added to the wash and bind solutions
    Failure to co-IP interacting proteinWash conditions were too stringent for the weak or transient interactionReduce the number of washes
    Lower the ionic strength of the wash buffer
    Interacting protein was expressed at a low levelApply additional protein sample
    Use a more sensitive detection system
    Buffer system was not optimal for the protein: protein interactionOptimize the co-IP buffer
    Insufficient sample was loaded on the gel for Western blot detectionElute sample in 30% acetonitrile 0.5% formic acid, then
    Bring the sample back up in SDS- PAGE Sample Buffer and load entire elution fraction on
    EphB3/HEK2/Eph Receptor B3 研究背景

    Ephrin type-B receptor 3, also known as EphB3 or HEK2, belongs to the ephrin receptor subfamily of the protein-tyrosine kinase family which 16 known receptors (14 found in mammals) are involved: EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6. The Eph family of receptor tyrosine kinases (comprising EphA and EphB receptors) has been implicated in synapse formation and the regulation of synaptic function and plasticity6. Ephrin receptors are components of cell signalling pathways involved in animal growth and development, forming the largest sub-family of receptor tyrosine kinases (RTKs). Ligand-mediated activation of Ephs induce various important downstream effects and Eph receptors have been studied for their potential roles in the development of cancer. EphB receptor tyrosine kinases are enriched at synapses, suggesting that these receptors play a role in synapse formation or function. We find that EphrinB binding to EphB induces a direct interaction of EphB with NMDA-type glutamate receptors. This interaction occurs at the cell surface and is mediated by the extracellular regions of the two receptors, but does not require the kinase activity of EphB.

    大鼠 EphB3/HEK2/Eph Receptor B3 參考資料
  • Bergemann AD, et al. (1998) Ephrin-B3, a ligand for the receptor EphB3, expressed at the midline of the developing neural tube. Oncogene. 16(4): 471-80.
  • Hock B, et al. (1998) PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor. Proc Natl Acad Sci U S A. 95(17): 9779-84.
  • Liu X, et al. (2006) EphB3: an endogenous mediator of adult axonal plasticity and regrowth after CNS injury. J Neurosci. 26(12): 3087-101.
  • Size / Price
    貨號: MB81593-T36-1
    目錄價: 
    單價:      (You Save: )
    規格:
    1 mL
    5 mL
    數量:+-
    庫存In Stock
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