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ONE-HOUR Western^TM Multiplex Kit II (β-Actin), 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 blots, either by cutting the membrane into half or by stripping the membrane after the first blot, to detect the target protein and the internal control separately. GenScript ONE-HOUR Western^TM Multiplex Kit II can detect both the target protein and β-actin in a single blot on the same membrane. Like other kits employing GenScript's ONE-HOUR Western^TM technology, it cuts blot time down to about one hour.

Using GenScript's immunodetection technology (patent pending), the kit replaces the classical three-step Western, which can take nearly five hours. To use the ONE-HOUR Western^TM Multiplex Kit II 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 II already contains a β-actin antibody that specifically reacts with human, mouse, rabbit, chicken, hamster, cow, goat, fish, and pig β-actin protein. This kit requires only a primary antibody for the target protein.

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

Low background: The kit contains WestClear^TM Nitrocellulose Membrane and LumiSensor^TM Plus 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.

No additional secondary antibody is needed: No extra β-actin antibody is needed.

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

Order:

Cat.No. Name Size Price Quantity   L00319 ONE-HOUR WesternTM Multiplex Kit II (β-Actin), Mouse  1 kit $ 345.00

Technical Manual: Download PDF file TM0256 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) PROTOCOL:  20080227011352  (PDF) Examples: 1. Shown below is a ONE-HOUR WesternTM Multiplex Blot performed using rabbit polyclonal antibody. The ONE-HOUR WesternTM Multiplex Kit II (β-Actin), Rabbit (GenScript, L00318) was used to detect both β-actin and GST protein (with the MW of about 26 kDa) in Hela cell lysate spiked with GST protein. Figure 1. Shown at right is a multiplex Western blot for the detection of both β-actin and GST protein from Hela cell lysate spiked with GST protein (MW of about 26 kDa) using the ONE-HOUR WesternTM Multiplex Kit II (β-Actin), Rabbit (GenScript, L00318) and Rabbit Anti-GST-tag Polyclonal Antibody (GenScript, A00097). 5 µg of Hela cell lysate (BD Biosciences, #611449) was spiked with 25.0, 12.5, 6.25, 3.13, 1.57 and 0 ng of Glutathione S-Transferase (GST), Schistosoma japonicum (GenScript, Z02039) 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 Multiplex WesternTM Blot performed using mouse monoclonal antibody. The ONE-HOUR WesternTM Multiplex Kit II (β-Actin), Mouse (GenScript, L00319) was used to detect both β-actin and GST protein (with the MW of about 26 kDa) in Hela cell lysate spiked with GST protein. Figure 2. Shown at right is a multiplex Western blot for the detection of both β-actin and GST protein from Hela cell lysate spiked with GST protein using the ONE-HOUR WesternTM Multiplex Kit II (β-Actin), Mouse (GenScript, L00319) and THETM Anti-GST Monoclonal Antibody (Mouse) (GenScript, A00865). 5 µg of Hela cell lysate (BD Biosciences, #611449) was spiked with with 25.0, 12.5, 6.25, 3.13, 1.57 and 0 ng of Glutathione S-Transferase (GST), Schistosoma japonicum (GenScript, Z02039) 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. In this case, 2 µg of THETM Anti-GST Monoclonal Antibody (Mouse) (GenScript, A00865) and 50 µl of WB-1 were mixed to make mixture 1. 3.Shown below is a ONE-HOUR Multiplex WesternTM Blot performed using goat polyclonal antibody. The ONE-HOUR WesternTM Multiplex Kit II (β-Actin), Goat (GenScript, L00320) was used to detect both β-actin and GAPDH protein in Hela cell lysate. Figure 3. Shown below is a multiplex Western blot for the detection of both β-actin and GAPDH from Hela cell lysate using the ONE-HOUR WesternTM Multiplex Kit II (β-Actin), Goat (GenScript, L00320) and Goat Anti-GAPDH Polyclonal Antibody (GenScript, A00191). 10, 5.0, 2.5, 1.25, and 0.63 µg of Hela cell lysate (BD Biosciences, #611449) were loaded into Lanes 1, 2, 3, 4, and 5, 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. In this case, 2 µg of Goat Anti-GAPDH Polyclonal Antibody (GenScript, A00191) and 50 µl of WB-1 were mixed to make mixture 1.

Related Products:   Code Name Size Price Quantity M01013  Solution: Pretreat A(100 ml) & B(100 ml) Solution  1 pk  $ 145.00 MB01011  Solution: 10X Wash Solution For One-Step Western Kits  100 ml  $ 65.00 L00225  LumiSensorTM Plus Chemiluminescent HRP Substrate Kit  1 Kit (2×30 ml)  $ 89.00 L00221V60  LumiSensorTM Chemiluminescent HRP Substrate Kit  1 Kit (2 X 30ml)  $ 55.00 L00224A60  WestClearTM Nitrocellulose Membrane  1 pk (10 sheets of 7.5 x 8 cm)  $ 60.50