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  • Immunoprecipitation (IP/CoIP) Kit
  • SKU: abs955
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    Product Details FAQ Citations(66) Video Pictures Documents
    Summary: Immunoprecipitation (IP/CoIP) is based on the specific affinity between antibody and protein. By capturing the antibody that binds to the protein or antigen, the target protein can be captured and enriched from a complex sample. At the same time, the proteins or other biomacromolecules interacting with the target protein can be measured.

    Before the experiment: In order to obtain better experimental results, all steps need to be optimized according to the actual situation.

    For example, more suitable cell lysis conditions may be selected depending on the cell line or subsequent use of the captured antigen. Mammalian cells without a cell wall structure can be cleaved directly with a stain remover, but other cells require some mechanical shear force, such as ultrasonic crushing. The conditions listed below (including lysates, incubation time, volume and concentration) are recommended as initial trials, and the final optimization needs to be adjusted according to the actual situation.

    In addition, the presence of the target protein in cell lysates can be determined by western blotting prior to immunoprecipitation.

    One, kit components
    The name of the reagentvolumePreservation conditions
    Lysis buffer30ml4 ℃
    10*Wash buffer50ml4 ℃
    1*SDS Sample buffer1ml4 ℃
    Protein A Agarose beads250ul4 ℃
    Protein GAgarose beads250ul4 ℃
    Note: 1 mM PMSF (YOU are recommended to purchase ABS9146 and prepare the Lysis Buffer by yourself) should be added immediately before use.

    2.use method

    A. Prepare cell lysates

    1. Blot the medium dry. A fresh medium containing regulatory molecules was added to allow the cells to be treated within a predetermined time.

    2. Cells were collected and washed once with 1 X PBS precooled with ice after removing the culture medium.

    3. Remove PBS and add 0.5 mL ice precooled Lysis buffer to each cell plate (10 cm) and incubate on ice for at least 5 minutes.

    4. Scrape the cells from the plate and transfer the extract to a microcentrifuge tube. On ice.

    5. Perform ultrasonic crushing on the ice for 3 times, 5 seconds each time.

    6. Microcentrifugation at 4 °C at 14,000 x g for 10 min. Transfer supernatant to new tube. The supernatant is the cell lysate. If necessary, the pyrolysis may be stored at -80 °C.

    Note: After the cell lysates are prepared, western blotting can be performed to determine the presence of the target protein in the cell lysates.

    B. Immunoprecipitation method

    A) Cell lysate pre-clearance (optional step)

    1. Add 5 μ L Protein A and 5 μ L Protein G to 500 μ L (containing 200-1000 ug total Protein) cell lysate.

    2. Rotate and incubate at 4 °C for 30-60 minutes.

    3. Centrifugation at 12000g at 4°C for 1 min. Reserve supernatant.

    B) antigen antibody binding

    1. Add 500 μ L (200 -- 1000 ug total protein) cell lysate (can be pre-cleaned cell lysate) to the new centrifuge tube.

    2. Add monoclonal/polyclonal antibody (1-5 μg) to target protein.

    3. Homologous antibodies of non-specific immunity were used as control.

    4. Blend gently at 4 °C overnight.

    C) Precipitation of immune complexes

    1. After incubation overnight, add 5 μ L Protein A and 5 μ L Protein G.

    2. Mix gently at 4 °C for 1-3 hours or overnight.

    3. Centrifuge at 12,000 g for 1 minute, retain the precipitation.

    4. Use 0.5 ml 1*Wash buffer to clean the precipitation, centrifuge at 12,000 g for 1 minute, and retain the precipitation.

    5. Repeat step 4 for a total of 3 times.

    Note: When cleaning and removing the supernatant, care should be taken to avoid suction of the filler

    C. Western blotting was used for analysis

    1. Suspend the precipitate in 20-40 μ L 1*SDS sample buffer, swirl the precipitate, then centrifuge for 30 seconds to remove beads and liquid from tube wall to tube bottom.

    2. Heat the sample to 95-100 °C for 2-5 minutes, then centrifuge at 14,000 g for 1 minute, remove supernatant.

    3. Load sample (15 -- 30 μ L) onto SDS-PAGE gel.

    4. Samples were analyzed by western blotting.

    3. Method of kit preservation

    The kit is stored at 4℃ and effective for 1 year.
    Tips:This product is for research use only. Not for use in diagnostic prodcedures.
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