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One-Step WesternTM Multiplex Kit I (GAPDH), Goat



Description:

Western blot analysis is one of the most common methods of detecting proteins and of determining specific protein concentrations in biological samples. However, loading errors and other human problems can confound this otherwise reliable system. An internal control is often needed to control and correct for such problems and to semi-quantitate protein expression levels. To date, the three most widely used internal controls are housekeeping proteins GAPDH, β-actin, and α-tubulin. However, to avoid crossover reactions, researchers usually need to perform two Western blot (by either cutting the membrane into two halves or by stripping the membrane after the first blot) to detect the target protein and the internal control separately. GenScript now introduces the One-Step WesternTM Multiplex Kit I. It can detect both the target protein and GAPDH in a single blot on the same membrane. Like other kits employing GenScript's One-Step WesternTM technology, it completes the blot in about one hour.

Using GenScript's breakthrough immunodetection technology (patent pending), the kit replaces the classical three-step Western, which can take nearly five hours. Transfer the proteins from gel to membrane and incubate it in the pretreat solution for five minutes. Then incubate in WB solution with primary antibody for 40 minutes, and, lastly, wash three times for ten minutes each. The membrane can then be developed with the HRP substrate included in the kit. The kit contains all the necessary reagents, buffers, nitrocellulose membrane and HRP substrate for performing a Western blot. The One-Step WesternTM Multiplex procedure is contrasted with a classical three-step Western at right.

The One-Step WesternTM Multiplex Kit I already contains a GAPDH antibody that specifically reacts with human, mouse, and rabbit GAPDH protein. Only a primary antibody for the target protein is required.

The One-Step WesternTM Multiplex Kit contains WestClearTM Nitrocellulose Membrane (0.2 µm) and LumiSensorTM Plus Chemiluminescent HRP substrate optimized for best results. WestClearTM Nitrocellulose Membrane and LumiSensorTM Plus Chemiluminescent HRP Substrate Kits are also available separately.

Key Features:

  • Easy to perform: The One-Step WesternTM Multiplex Kit has fewer and simpler steps than other Western kits, leaving fewer chances for human error.
  • Low background: The One-Step WesternTM Multiplex Kit contains WestClearTM Nitrocellulose Membrane and LumiSensorTM Plus Chemiluminescent HRP Substrate Kit, optimized for low background.
  • High sensitivity: The One-Step WesternTM Multiplex Kit is 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 One-Step WesternTM Multiplex Kit produces highly reproducible results.
  • No secondary antibody is needed: No extra GAPDH antibody is needed.
  • The One-Step WesternTM Multiplex Kit needs far less optimization than the classical three-step method.
    Order:

     Cat.No.NameSizePriceQuantity
     L00317 One-Step WesternTM Multiplex Kit I (GAPDH), Goat  1 kit$ 345.00


    Technical Manual:

    Components:
    ComponentsL00315L00316L00317L00319L00320L00321
    Pretreat A Solution 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 
    Pretreat B Solution 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 
    WB-1 Solution 1ml 1ml 1ml 1ml 1 ml 1 ml 
    WB-2 Solution 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 
    10 X Wash Solution 125 ml 125 ml 125 ml 125 ml 125 ml 125 ml 
    WestClearTM Nitrocellulose Membrane 10 sheets 10 sheets 10 sheets 10 sheets 10 sheets 10 sheets 
    LumiSensorTM Plus Chemiluminescent 2 X 30 ml 2 X 30 ml 2 X 30 ml 2 X 30 ml 2 X 30 ml 2 X 30 ml 
    Protocol 1 1 1 1 1 1 

    PROTOCOL:  20071101145526  (PDF)
    MSDS:  20071207115010  (PDF)


    Examples:

    1.Shown below is a One-Step WesternTM Multiplex Blot performed using rabbit polyclonal antibody.
    The One-Step WesternTM Multiplex Kit I (GAPDH), Rabbit (GenScript, L00315) was used to detect both GAPDH and a His-tagged protein (with the MW of about 45 Kd) in Hela cell lysate spiked with a His-tagged protein.
    Figure 1. Shown at right is a multiplex Western blot for the detection of both GAPDH and a His-tagged protein from Hela cell lysate spiked with a His-tagged protein (MW of about 45 Kd) using the One-Step WesternTM Multiplex Kit I (GAPDH), Rabbit (GenScript, L00315) and Rabbit Anti-His-tag Polyclonal Antibody (GenScript, A00174). 5 µg of Hela cell lysate (BD Biosciences, #611449) was spiked with 50.0, 25.0, 12.5, 6.25, 3.13, and 0 ng of a His-tagged protein and loaded into Lanes 1, 2, 3, 4, 5, and 6, respectively. The Western blot was performed following the manufacturer's protocol and developed with the LumiSensorTM Plus Chemiluminescent HRP Substrate Kit (GenScript, L00225) included in the kit.
    One-Step Western<sup>TM</sup> Multiplex Blot performed using rabbit polyclonal antibody

    2.Shown below is a One-Step WesternTM Multiplex Blot performed using mouse monoclonal antibody.
    The One-Step WesternTM Multiplex Kit I (GAPDH), Mouse (GenScript, L00316) was used to detect both GAPDH and Trx protein in Hela cell lysate spiked with Trx protein.
    Figure 2.Shown at right is a multiplex Western blot for the detection of both GAPDH and Trx protein from Hela cell lysate spiked with Trx protein using the One-Step WesternTM Multiplex Kit I (GAPDH), Mouse (GenScript, L00316) and Mouse Anti-Trx-tag Monoclonal Antibody (GenScript, A00180). 5 µg of Hela cell lysate (BD Biosciences, #611449) was spiked with 50.0, 25.0, 12.5, 6.25, 3.13, and 0 ng of Trx protein and loaded into Lanes 1, 2, 3, 4, 5, and 6, respectively. The Western blot was performed following the manufacturer's protocol and developed with the LumiSensorTM Plus Chemiluminescent HRP Substrate Kit (GenScript, L00225) included in the kit.
    One-Step Multiplex Western<sup>TM</sup> Blot performed using mouse monoclonal antibody

    3.Shown below is a One-Step Multiplex WesternTM Blot performed using goat polyclonal antibody.
    The One-Step WesternTM Multiplex Kit I (GAPDH), Goat (GenScript, L00317) was used to detect both GAPDH and Trx protein in Hela cell lysate spiked with Trx protein. Figure 3. Shown below is a multiplex Western blot for the detection of both GAPDH and Trx protein from Hela cell lysate spiked with Trx protein using the One-Step WesternTM Multiplex Kit I (GAPDH), Goat (GenScript, L00317) and Goat Anti-Trx-tag Polyclonal Antibody (GenScript, A00129). 5 µg of Hela cell lysate (BD Biosciences, #611449) was spiked with 50.0, 25.0, 12.5, 6.25, 3.13, and 0 ng of Trx protein and loaded into Lanes 1, 2, 3, 4, 5, and 6, respectively. The Western blot was performed following the manufacturer's protocol and developed with the LumiSensorTM Plus Chemiluminescent HRP Substrate Kit (GenScript, L00225) included in the kit.
    One-Step Multiplex Western<sup>TM</sup> Blot performed using goat polyclonal antibody


    Related Products:

      Code NameSizePriceQuantity
    L00315  One-Step WesternTM Multiplex Kit I (GAPDH), Rabbit 1 kit 
    $ 345.00
    L00316  One-Step WesternTM Multiplex Kit I (GAPDH), Mouse 1 kit 
    $ 345.00
    L00224A60  WestClearTM Nitrocellulose Membrane 1 pk (10 sheets of 7.5 x 8 cm) 
    $ 60.50
    L00221V60  LumiSensorTM Chemiluminescent HRP Substrate Kit 1 Kit (2 X 30ml) 
    $ 55.00
    L00225  LumiSensorTM Plus Chemiluminescent HRP Substrate Kit 1 Kit (2×30 ml) 
    $ 89.00
    M01013 Solution: Pretreat A(100 ml) & B(100 ml) Solution 1 pk 
    $ 145.00
    MB01011 10X Wash Solution For One-Step Western Kits 100 ml 
    $ 65.00



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