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This product has been discontinued!

ONE-HOUR Western^TM Multiplex Kit I (GAPDH), Mouse

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-HOUR Western^TM 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-HOUR Western^TM 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-HOUR Western^TM Multiplex procedure is contrasted with a classical three-step Western at right.

The ONE-HOUR Western^TM 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-HOUR Western^TM Multiplex Kit contains WestClear^TM Nitrocellulose Membrane (0.2 µm) and LumiSensor^TM Plus Chemiluminescent HRP substrate optimized for best results. WestClear^TM Nitrocellulose Membrane and LumiSensor^TM Plus Chemiluminescent HRP Substrate Kits are also available separately.

Key Features:

Easy to perform: The ONE-HOUR Western^TM Multiplex Kit has fewer and simpler steps than other Western kits, leaving fewer chances for human error.

Low background: The ONE-HOUR Western^TM Multiplex Kit contains WestClear^TM Nitrocellulose Membrane and LumiSensor^TM Plus Chemiluminescent HRP Substrate Kit, optimized for low background.

High sensitivity: The ONE-HOUR Western^TM 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-HOUR Western^TM Multiplex Kit produces highly reproducible results.

No additional secondary antibody is needed: No extra GAPDH antibody is needed.

The ONE-HOUR Western^TM Multiplex Kit needs far less optimization than the classical three-step method.

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Cat.No. Name Size Price Quantity

Technical Manual: Download PDF file TM0255 Components: Components L00315 L00316 L00317 L00318 L00319 L00320 L00321 L00322 L00323 Pretreat A Solution  100 ml  100 ml  100 ml  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  100 ml  100 ml  100 ml  WB-1 Solution  1ml  1ml  1ml  1ml  1ml  1 ml  1 ml  1 ml  1 ml  WB-2 Solution  100 ml  100 ml  100 ml  100 ml  100 ml  100 ml  100 ml  100 ml  100 ml  10X Wash Solution  125 ml  125 ml  125 ml  125ml  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  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  2 x 30 ml  2 x 30 ml  2 x 30 ml  Protocol  1  1  1  1  1  1  1  1  1  MSDS:  20071207115010  (PDF) Examples: 1.Shown below is a ONE-HOUR WesternTM Multiplex Blot performed using rabbit polyclonal antibody. The ONE-HOUR 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-HOUR 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. 2.Shown below is a ONE-HOUR WesternTM Multiplex Blot performed using mouse monoclonal antibody. The ONE-HOUR 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-HOUR 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. 3.Shown below is a ONE-HOUR Multiplex WesternTM Blot performed using goat polyclonal antibody. The ONE-HOUR 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-HOUR 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.

Related Products:   Code Name Size Price Quantity M01013  Solution: Pretreat A(50 ml) & B(50 ml) Solution  1 pk  $ 30.00 L00221V60  LumiSensorTM Chemiluminescent HRP Substrate Kit  1 Kit (2 X 30ml)  $ 55.00 L00224A60  WestClearTM Nitrocellulose Membrane  1 pk (5 sheets of 7.5 x 8 cm)  $ 20.00 MB01011  Solution: 5X Wash Solution For One-Step Western Kits  125 ml  $ 28.00

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