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Immunoprecipitation/IP Kit-Immunomagnetic Beads Protein A/G

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    Product Content
     
    Contents BAG001-20 BAG001-100 Storage
    Immunomagnetic Beads Protein A/G[1] 1 mL 5 mL 2-8℃ for 12 months
    1×PBST (pH=7.4) Required but not supplied  
    1×PBS (pH=7.4) Required but not supplied  
    NP40 Cell Lysis Buffer2 4 mL 20 mL -20℃ for 12 months
    Elution Buffer 1.5 mL 8 mL 2-8℃ for 12 months
    Neutralization Buffer 1.5 mL 8 mL 2-8℃ for 12 months

    [1] Immunomagnetic Beads Protein A/G (1:1) contains 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
     
    Protein A/G
    Product Description
     

    The key component, Immunomagnetic Beads Protein A/G is prepared using recombinant Protein A/G (10600-P07E/(13103-PNAE, Sino Biological Inc.).
    The Immunomagnetic Beads Protein A/G is designed for Immunoprecipitation / IP of proteins, protein complex, protein-nucleic acid complex, and other antigens.
    Your antibody is added to a tube containing Immunomagnetic Beads Protein A/G. After a short incubation, the antibody's Fc-region binds to the Protein A/G. The tube is then placed on a Magnetic Separator, where the Immunomagnetic Beads migrate to the side wall of the tube facing the Magnetic Separator and allow for easy removal of the supernatant.
    The Immunomagneatic Beads-bound antibodies can now be used for IP. Bound material is easily collected using the unique magnetic properties of the Immunomagnetic Beads.

    IP Experimental results
     
    Protein A/G
    Items Lane

    Cell sample (0.5mg)

    A B

    Hela

    293T
    Polyclonal antibody (2 μL) GAPDH Antibody, Rabbit PAb, Antigen Affinity Purified (10094-T52)
    Beads Protein A/G Immunomagnetic beads-100 μL
    Primary antibody GAPDH Antibody, Rabbit PAb, Antigen Affinity Purified (10094-T52) at 10 μg/mL
    Secondary antibody Dylight 800-labeled antibody to rabbit IgG (H+L), at 1:7500 dilution
    Gel 13% SDS-PAGE reducing gel
    Protocol
     
    Protein A
    Fig. 1 Immunoprecipitation (IP) Protocol

    The protocol (Fig. 1) uses 50 μL Immunomagnetic Beads Protein A, 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).

    Prepare Beads
    1. Resuspend Immunomagnetic Beads in the vial (vortex >30 sec or tilt and rotate 5 min).
    2. Transfer 50 μL Immunomagnetic Beads to a tube.
    3. Place the tube on the Magnetic Separator to separate the Immunomagnetic Beads from the solution, and remove the supernatant.
    4. Remove the tube from the Magnetic Separator.
    Bind Antibody
    1. Dilute your antibody (typically 1–10 μg) in 200 μL PBST and add to the Immunomagnetic Beads. The amount of antibody used needs to be optimized.
    2. Incubate and rotate for 10 min at room temperature.
    3. Place the tube on the Magnetic Separator and remove the supernatant.
    4. Remove the tube from the Magnetic Separator and wash the Immunomagnetic Beads using 200 μL PBST, by gentle pipetting.
    For storage of antibody-conjugated Immunomagnetic Beads, we suggest to use PBST to prevent aggregation.
    Immunoprecipitate Target Antigen
    1. Place the tube (from Bind Antibody step 5) on the Magnetic Separator and remove the supernatant.
    2. Add your sample (typically 100–1,000 μL) to the tube and gently mix the Immunomagnetic Beads-antibody complex by gentle pipetting
    3. Incubate with rotation for 10 min at room temperature to allow antigen to bind to the Immunomagnetic Beads-antibody complex.
    Note: it may be necessary to increase incubation times for optimal binding.
    4. Place the tube on the Magnetic Separator and remove the supernatant or transfer the supernatant to a clean tube for further analysis, if desired.
    5. Wash the Immunomagnetic Beads-antibody-antigen complex 3 times using 200 μL PBS for each wash. Separate on the Magnetic Separator between each wash, remove the supernatant and resuspend the complex by gentle pipetting.
    6. Resuspend the above complex in 100 μL PBS and transfer the Immunomagnetic Beads suspension to a clean tube. This is recommended to avoid co-elution of proteins bound to the tube wall.
    Elute Target Antigen
    A. Denaturing Elution
    1. Place the tube (from step 6 in Immunoprecipitation of Target Antigen) on the Magnetic Separator and remove the supernatant.
    2. Add 20 μL Elution Buffer and 5 μL 5×SDS-PAGE Sample Buffer and resuspend the Immunomagnetic Beads complex by gently pipetting.
    3. Heat for 5-10 min at 95-100 ℃.
    4. Place the tube on the Magnetic Separator and collect the supernatant for further analysis.
    B. Mild Elution
    1. Place the tube (from step 6 in Immunoprecipitation of Target Antigen) on the Magnetic Separator and remove the supernatant.
    2. Add 20 μL Elution Buffer and gently pipette to resuspend the complex. Avoid foaming.
    3. Incubate with rotation for 2 min at room temperature to dissociate the complex.
    4. Place the tube on the Magnetic Separator and transfer the supernatant containing eluted antibody and antigen to a clean tube.
    If the eluted protein is to be used for functional assays or stored, the pH of the eluate can be adjusted by adding Neutralization Buffer (about 50µL of Neutralization Buffer for each 100µL Elution buffer).
    Target Antigen Detection
    A. Denaturing Elution
    1. SDS-PAGE for staining and protein identification
    2. SDS-PAGE for Western blotting
    3. SDS-PAGE for Fluorography
    B. Mild Elution
    1. Protein characterization
    2. Immunization
    3. Enzyme studies
    4. AA sequence determination
    5. Crystallization
    C. No Elution
    1. Protein interaction
    2. Enzyme studies
    3. Bioassays

    Reference Information
     

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    Binding Characteristics of Protein A/G/L

    Native Protein A/G/L differ in their binding to Igs from different species and subclasses. For example, human IgG3 will bind strongly to Protein G, but weakly to Protein A.

    Species

    Protein A

    Protein G

    Protein L

     Human

     IgG

    +++

    +++

    +++

     

     IgG1

    ++++

    ++++

    ++++

     

     IgG2

    ++++

    ++++

    ++++

     

     IgG3

    -

    +++

    +++

     

     IgG4

    ++++

    ++++

    ++++

     

     IgA

    +

    -

    +++

     

     IgA1

    +

    -

    +++

     

     IgA2

    +

    -

    +++

     

     IgD

    -

    -

    +++

     

     IgE

    ++

    -

    +++

     

     IgM

    +

    -

    +++

     Rabbit

     IgG

    +++

    +++

    +

     Cow

     IgG

    +

    +++

    -

     

     IgG1

    +

    +++

    -

     

     IgG2

    +++

    +++

    -

     Cat

     IgG

    +++

    +

    ?

     Horse    

     IgG

    ++

    ++++

    ?

     Goat

     IgG

    +

    ++

    -

     

     IgG1

    +

    +++

    -

     

     IgG2

    +++

    +++

    -

     Guinea-pig

     IgG1

    ++

    +

    ?

     

     IgG2

    ++

    +

    ?

     Sheep

     IgG

    +

    ++

    -

     

     IgG1

    +

    ++

    -

     

     IgG2

    +++

    +++

    -

     Dog

     

    ++

    +

    ?

     Pig

     

    +++

    ++

    +++

     Rat

     IgG

    +

    ++

    +++

     

     IgG1

    -

    +

    +++

     

     IgG2a

    -

    ++++

    +++

     

     IgG2b

    -

    ++

    +++

     

     IgG2c

    ++

    ++

    +++

     

     IgG3

    +

    ++

    ?

     Mouse

     IgG

    ++

    ++

    +++

     

     IgG1

    +

    ++++

    +++

     

     IgG2a

    ++++

    ++++

    +++

     

     IgG2b

    +++

    +++

    +++

     

     IgG3

    ++

    +++

    +++

     

     IgM

    -

    -

    +++

     Chicken

     IgY

    -

    -

    -

     Monkey(rhesus)

     IgG

    ++++

    ++++

    ?

     Hamster

     

    +

    ++

    +++

     Koala

     

    -

    +

    ?

     Llama

     

    -

    +

    ?

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    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"