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One-Step WesternTM Multiplex Kit III (α-Tubulin), 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 blots (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 III. It can detect both the target protein and α-Tubulin 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 III already contains a α-Tubulin antibody that specifically reacts with human, mouse, rabbit and rat α-Tubulin protein. Only a primary antibody for the target protein is required.
The 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: This kit has fewer and simpler steps than other Western kits, leaving fewer chances for human error.
Low background: The kit contains WestClearTM Nitrocellulose Membrane and LumiSensorTM 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 secondary antibody is needed: No extra α-Tubulin antibody is needed. The One-Step WesternTM needs far less optimization than the classical three-step method.
1. Shown below is a One-Step Multiplex WesternTM Blot performed using rabbit polyclonal antibody. The One-Step WesternTM Multiplex Kit III (a-Tubulin), Rabbit (GenScript, L00321) was used to detect both α-Tubulin and Trx protein (with the MW of about 12 kDa) in Hela cell lysate spiked with Trx protein. Figure 1. Shown below is a multiplex Western blot for the detection of both α-Tubulin and Trx protein from Hela cell
lysate spiked with Trx protein (MW of about 12 kDa) using the One-Step WesternTM Multiplex Kit III (a-Tubulin), Rabbit (GenScript, L00321) and rabbit polyclonal antibody against Trx (Sigma, T0803). 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.
2.Shown below is a One-Step Multiplex WesternTM Blot performed using mouse monoclonal antibody. The One-Step WesternTM Multiplex Kit III (a-Tubulin), Mouse (GenScript, L00322) was used to detect both α-Tubulin and GST protein (with the MW of about 26 kDa) in Hela cell lysate spiked with GST protein. Figure 2. Shown below is a multiplex Western blot for the detection of both α-Tubulin and GST protein from Hela cell lysate spiked with GST protein using the One-Step WesternTM Multiplex Kit III (a-Tubulin), Mouse (GenScript, L00322) 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, 1.56, 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-Step Multiplex WesternTM Blot performed using goat polyclonal antibody. The One-Step WesternTM MultiplexKit III (a-Tubulin), Goat (GenScript, L00323) was used to detect both α-Tubulin and GAPDH protein in Hela cell lysate. Figure 3. Shown below is a multiplex Western blot for the detection of both α-Tubulin and GAPDH from Hela cell lysate using the One-Step WesternTM MultiplexKit III (a-Tubulin),Goat (GenScript, L00323) and Goat Anti-GAPDH Polyclonal Antibody (GenScript, A00191). 5.0, 2.5, 1.3, 0.63 and 0.31 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.
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 (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 III. It can detect both the target protein and α-Tubulin 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. 
