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Immunoprecipitation/IP Kit- HA Tag Immunomagnetic Beads

    產品資料評論相關產品實驗方法
    Product Content
     
    Contents TB100028-20 TB100028-100 Storage
    HA Tag Immunomagnetic Beads1 1 mL 5 mL 2-8℃ for 12 months
    NP40 Cell Lysis Buffer2 4 mL 22 mL -20℃ for 12 months
    5×TBST(pH7.4) Required but not supplied  
    1×TBST(pH7.4) Required but not supplied  
    ddH2O Required but not supplied  
    HA Tag Positive Cell Lysis 300 μg 300 μg -20℃ for 12 months
    Alkaline Elution Buffer 3 mL 15 mL 2-8℃ for 12 months
    Acidity Elution Buffer 3 mL 15 mL 2-8℃ for 12 months
    Neutralization Buffer 2 mL 8 mL 2-8℃ for 12 months
    HA Synthetic Peptide Not included (refer related product PP100028) Not included (refer related product PP100028) -20℃ for 12 months

    [1] HA Tag Immunomagnetic Beads contain immunomagnetic beads (2 mg/mL) in phosphate buffered saline (PBS, pH 7.4) with sodium azide (0.1%).
    [2] Using NP-40 cell lysate buffer in the kit is required,otherwise,the magnetic beads may be precipitated.

    Product Image
     
    HA
    Product Description
     

    The HA Tag Immunomagnetic Beads, conjugated with HA tag Antibody (100028-MM15), are used for Immuneprecipitation / IP of HA-tagged proteins expressed in vitro expression systems. For IP, the Immunomagnetic Beads are added to a sample containing HA-tagged proteins to form a bead-protein complex. The complex is removed from the solution manually against a Magnetic Separator. The bound HA-tagged proteins are dissociated from the Immunomagnetic Beads using an Elution Buffer.

    Antibody Information
     

    Antibody: HA Tag Antibody, Mouse MAb (100028-MM15)
    Immunogen: A synthetic peptide corresponding to the HA-tag sequence.
    Clone ID: MM15
    Isotype: IgG
    Specificity: Recognize N-terminal and C-terminal HA Tag in fusion proteins.
    Preparation: This antibody was produced from a hybridoma resulting from the fusion of a mouse myeloma with B cells obtained from a mouse immunized with purified, a synthetic peptide corresponding to the HA-tag sequence. The IgG fraction of the cell culture supernatant was purified by Protein A affinity chromatography.

    IP Experimental results
     
    HA
    Items Lane

    Sample (30 μg)

    (Whole cell lysate)

    A B C
    HA-ARG1-myc Transfected 293 myc-ARG1-HA Transfected 293 pSTEP2 Transfected 293
    Beads SBI Anti-HA Tag Immunomagnetic Beads-30 μL
    WB detection antibody Anti-HA Tag Antibody, Mouse Mab (100028-MM10) at 1 μg/mL
    Gel 13% SDS-PAGE reducing gel
    Secondary antibody Dylight 800-labeled antibody to mouse IgG (H+L), at 1:5000 dilution
    Protocol
     
    HA
    Fig. 1 Immunoprecipitation (IP) Protocol

    The protocol (Fig. 1) uses 50 μL HA Tag 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). Add prtease inhibitor (such as PMSF at 1mM) if needed.
    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 Immunomagnetic Beads against the side wall 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 the 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 Beads on a Magnetic Separator and discard the supernatant.
    Elute Target Antigen
    A. Neutral Elution Protocol
    1. Prepare HA peptide (PP100028) at 1mg/mL in PBS.
    2. Add 50 μL 1 mg/mL HA peptide to the Immunomagnetic Beads, gently vortex to mix and incubate the sample at 37 ℃ on a rotator for 5-10 min. Elution may be performed at reduced temperatures, but lower yields may result.
    3. Separate the Immunomagnetic beads on a Magnetic Separator and save the supernatant containing the target antigen.
    4. Repeat Elution step once for higher recovery.
    B. Alkaline Elution Protocol
    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.
    C. Acidity Elution Protocol
    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.
    D. 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 95-100℃ for 5-10 min.
    3. Magnetically separate the Immunomagnetic Beads and save the supernatant containing the antigen.
    Usage of positive cell lysate
    When detecting the antigen with western blotting, the positive cell lysates (supplied in the KIT) can be used as a positive western blotting cell lysates sample.
    Note: For tag magnetic beads the positive cell lysates also can be used as a IP cell lysates sample.

    Reference Information
     

    Related Products

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    Immunoprecipitation Kit -Immunomagnetic Beads Protein L BL11044
    Immunoprecipitation Kit -Immunomagnetic Beads Protein A/G BAG001
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    Trouble Shooting

    Problem Possible Cause Solution
    Little or no HA-tagged protein is detected Tagged protein degraded Include protease inhibitors (PMSF) in the lysis buffer
    Use new lysate or lysate stored at -80°C
    No or minimal tagged protein was expressed Verify protein expression by SDS-PAGE or Western blot
    analysis of the lysate using an HA-tagged positive control as a reference
    Increase the amount of lysate used for IP/Co-IP
    Use a more sensitive detection system
    Magnetic Beads aggregated Magnetic Beads were frozen or centrifuged Handle the Beads as directed in the instructions
    Buffer was incompatible with magnetic beads
    Detergent was not added to the wash and bind solutions
    Failure to co-IP interacting protein Wash conditions were too stringent for the weak or transient interaction Reduce the number of washes
    Lower the ionic strength of the wash buffer
    Interacting protein was expressed at a low level Apply additional protein sample
    Use a more sensitive detection systemds
    Buffer system was not optimal for the protein: protein interaction Optimize the co-IP buffer
    Insufficient sample was loaded on the gel for Western blot detection Elute 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
    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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