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Immunoprecipitation/ IP Kit - Anti-CD166 / ALCAM Immunomagnetic Beads

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    Immunoprecipitation/ IP Kit- Anti-CD166 / ALCAM Immunomagnetic Beads
    Product Contents
    ContentsPackage 1Package 2Storage
    Anti-CD166/ALCAM 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
    CD166/ALCAM 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-CD166/ALCAM 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-CD166/ALCAM Immunomagnetic Beads, conjugated with Anti-CD166/ALCAM antibody, are used for immuneprecipitation (IP) of CD166/ALCAM proteins which expressed in vitro expression systems and bacterial and mammalian cell lysates. For IP, the beads are added to a sample containing CD166/ALCAM proteins to form a bead-protein complex. The complex is removed from the solution manually using a Magnetic Separator. The bound CD166/ALCAM proteins are dissociated from the Immunomagnetic Beads using an elution buffer.

    Antibody Information
    Antibody:90227-T52
    Immunogen:Recombinant Cynomolgus CD166 / ALCAM protein (Catalog#90227-C08H)
    Isotype:Rabbit IgG
    分子種屬:Cynomolgus
    产品或服务:1
    Clone ID:
    Molecule:ALCAM
    抗體製備方法:Produced in rabbits immunized with purified, recombinant Cynomolgus CD166 / ALCAM (rh CD166 / ALCAM; Catalog#90227-C08H; G7NZQ8; Met1-Ala526). CD166 / ALCAM specific IgG was purified by Cynomolgus CD166 / ALCAM affinity chromatography.
    特異性:Cynomolgus CD166 / ALCAM
    Guaranteed Applications:IP,Minimum Protein Purification
    IP Protocol

    Fig. 1 Immunoprecipitation (IP) Protocol

    The protocol (Fig. 1) uses 50 μL Anti-CD166/ALCAM 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
    CD166/ALCAM 研究背景

    Activated leukocyte cell adhesion molecule (ALCAM)/Cluster of differentiation (CD166) is a type I transmembrane cell adhesion molecule belonging to the Ig superfamily and a ligand for CD6 that is expressed on T lymphocytes. The extracellular domain of ALCAM contains five Ig-like domains (three Ig-like C2-type domains and two Ig-like V-type domains), of which the amino-terminal V1 domain is essential for ligand binding and ALCAM-mediated cell aggregation. ALCAM mediates both heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. ALCAM/CD6 interaction plays a role in T cell development and T cell regulation, as well as in the binding of T- and B-cells to activated leukocytes. Recently, homophilic (ALCAM-ALCAM) adhesion was shown to play important roles in tight cell-to-cell interaction and regulation of stem cell differentiation. While expressed in a wide variety of tissues, ALCAM is usually restricted to subsets of cells involved in dynamic growth and/or migration, including neural development, branching organ development, hematopoiesis, immune response and tumor progression. And CD166 is regarded as a potential novel breast cancer indicator and therapeutic target.

    食蟹猴 CD166/ALCAM 參考資料
  • Swart GW. (2002) Activated leukocyte cell adhesion molecule (CD166/ALCAM): developmental and mechanistic aspects of cell clustering and cell migration. Eur J Cell Biol. 81(6): 313-21.
  • Fujiwara H, et al. (2003) Human blastocysts and endometrial epithelial cells express activated leukocyte cell adhesion molecule (ALCAM/CD166). J Clin Endocrinol Metab. 88(7): 3437-43.
  • Jezierska A, et al. (2006) ALCAM/CD166 protects breast cancer cells against apoptosis and autophagy. Med Sci Monit. 12(8): BR263-73.
  • Kahlert C, et al. (2009) Increased expression of ALCAM/CD166 in pancreatic cancer is an independent prognostic marker for poor survival and early tumour relapse. Br J Cancer. 101(3): 457-64.
  • All information of our products is subject to change without notice. Please refer to COA enclosed in shipped package for the newest information.
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