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One-Step IP-Western Kit (Mouse)


Kit Description:


GenScript One-Step IP-Western Kit contains all the necessary reagents, buffers, nitrocellulose membrane and HRP substrate for performing an IP-Western blot. It was developed using breakthrough immuno-detection technology (patent pending), which allows the user to perform a complete IP-Western blot in a fraction of the usual time. After transferring the proteins from gel to membrane, simply incubate the membrane in Pretreat Solution for five minutes and follow with incubation in WB solution (prepared from primary antibody, IP-WB 1, IP-WB 2 and IP-WB 3) for 40 minutes. Wash the membrane three times for five minutes each and then the blot can be developed using the LumiSensorTM Chemiluminescent HRP Substrate Kit included in the kit.

The One-Step IP-Western Kit (L00232) with mouse antibody allows for the detection of nanograms of antigen while leaving quantities of mouse primary antibody (or any other antibody) of up to 2 µg per lane undetected, giving a clearer, more useful result. With this kit, protein G blocker is only needed if protein G or A/G resin is used during immunoprecipitation. Protein A carryover does not affect the western results.

The results of the One-Step IP-Western blot result are compared to results of a conventional three-step Western in figure 1 at right.


Key Features:

  • Easy to perform: Fewer steps mean fewer chances for human error.
  • Compatible with immunoprecipitation methods incorporating protein A, G, or A/G resin or any secondary antibody.
  • Low background: The kit contains WestClearTM Nitrocellulose Membrane and LumiSensorTM Chemiluminescent HRP Substrate Kit, optimized for low background.
  • High sensitivity: The kit's sensitivity is comparable to or better than that of the classical 4.5-hour procedure, depending on the quality and amount of antibodies used.
  • Reproducible results: The kit produces highly reproducible results.
  • Needs far less optimization than the classical three-step method.
  • No secondary antibody is needed.

    Applications:

    The One-Step IP-Western Kit enables fast the Western and Dot blots for the detection of proteins from immunoprecipitation. It is compatible with the following resins:
  • Protein A, G, or A/G resin
  • Any secondary antibody resin

    Order:

     Cat.No.NameSizePriceQuantity
     L00232 One-Step IP-Western Kit (Mouse)  1 Kit (10 Assays)$ 375.00

    Technical Manual:

    Components:
    Components L00231(Rabbit)L00232(Mouse)L00233(Goat)
    Pretreat A solution 100 ml 100 ml 100 ml 
    Pretreat B solution 100 ml 100 ml 100 ml 
    A&G blocker (100X)  1 ml 1 ml 
    Protein G blocker (100X) 1 ml   
    IP-WB 1 solution 1 ml 1 ml 1 ml 
    IP-WB 2 solution 1 ml 1 ml 1 ml 
    IP-WB 3 solution 100 ml 100 ml 100 ml 
    10X Wash solution 125 ml 125 ml 125 ml 
    WestClearTM Nitrocellulose Membrane (0.2 µm, 7.5 x 8 cm) 10 Sheets 10 Sheets 10 Sheets 
    LumiSensorTM Chemiluminescent HRP Substrate 2 x 30 ml 2 x 30 ml 2 x 30 ml 
    Protocol 1 1 1 

    PROTOCOL:  20061201105101  (PDF)
    MSDS:  20061201152646  (PDF)



    Examples:


    Western blot detection using mouse primary antibody.
    Western blots for the detection of multiple tag fusion protein using Mouse Anti Trx-Tag mAb (GenScript, A00180) by both classical Western and One-Step IP-Western (using kit L00232). Both blots were developed with the LumiSensorTM Chemiluminescent HRP Substrate included in kit L00232.

    Figure 3. Western blot detection of multiple-tag fusion protein by both classical western and One-StepTM IP-Western (using kit L00232). Both blots are developed using the LumiSensorTM Chemiluminescent HRP Substrate that is included in kit L00232
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