One-Step Western^TM Advanced Kit

Kit Description:

The Advanced Western Blot Kit is designed to result in high sensitivity and low background as compared to traditional Western blot analysis. GenScript One-Step Western^TM Advanced kits reveal even the tiniest amounts of relevant product. Do not let an inferior Western result overlook small but significant results. Like our standard One-Step Western^TM, the Advanced Western cuts western and dot blot time significantly. However, the One-Step Western^TM Advanced boasts even greater sensitivity and lower background than our standard kit.

One-Step Western^TM Advanced Kit contains all the necessary reagents, buffers, nitrocellulose membrane and HRP substrate for performing a Western Blot. It was developed using breakthrough immuno-detection technology (patent pending), allowing the user to perform a complete 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 5 minutes and follow with incubation in WB solution (prepared from primary antibody, WB-1 and WB-2) for 40 minutes. Wash the membrane three times for 10 minutes each and then the blot can be developed using the HRP substrate included in the kit. The One-Step Western^TM Advanced Blot procedure is compared to the classical three-step procedure in Figure 1.

The 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: Simple and rapid procedure in less than 1.5 hours, and no secondary antibody is needed.
• Low background: Faster, surer results with journal-quality images.
• High sensitivity: Sensitivity is higher than that of the classical 4.5-hour procedure, depending on the quality and quantity of antibodies used.

Storage:

Store WestClear^TM Nitrocellulose Membrane at room temperature. Store the rest of the kit at 4°C. It will remain stable for six months. Do not freeze the kit or any component.

Technical Manual:

• Download PDF file TM0352

Application Examples:

Comparison of the three One-Step Western^TM Blot Kits of different sensitivities using monoclonal antibodies:
Two similar blots were processed with the same procedures using different One-Step Western^TM Kits: Standard (L00205C) and Advanced (L00242). 10 µg and 2.5 µg of GenScript Mouse Monoclonal Anti-GST (GenScript, A00865), respectively, were used with these two kits to detect GST protein.
The results are shown in Figure 2.

Figure 2. Western blots for the detection of GST protein by One-Step Western^TM using different One-Step Western^TM Kits: Standard (L00205C) and Advanced (L00242). 33.0 11.0, 3.60, 1.20 0.41 0.13, and 0.045 ng of GST protein were loaded into Lanes 1, 2, 3, 4, 5, 6, and 7, respectively.

Comparison of the three One-Step Western^TM Blot Kits of different sensitivities using polyclonal antibodies:
Two similar blots were processed with the same procedures using different One-Step Western^TM Kits: Standard (L00204C) and Advanced (L00241). 10 µg, 10 µg, and 2.5 µg of GenScript Rabbit Polyclonal Anti-GST (GenScript, A00097), respectively, were used with the three kits to detect GST protein.
The results are shown in Figure 3.

Figure 3. Western blots for the detection of GST protein by One-Step Western^TM using different One-Step Western^TM Kits: Standard (L00204C) and Advanced (L00241). 25.0 12.5, 6.25, 3.10, 1.50, and 0.78 ng of GST protein were loaded into Lanes 1, 2, 3, 4, 5, and 6, respectively.