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 TM0223

Components:

Components	L00241	L00242	L00243
Application	Quick and Sensitive Western Detection (Rabbit)	Quick and Sensitive Western Detection (Mouse)	Quick and Sensitive Western Detection (Goat)
Pretreat A Solution	100 ml	100 ml	100 ml
Pretreat B Solution	100 ml	100 ml	100 ml
WB-1 Solution	1 ml	1 ml	1 ml
WB-2 Solution	100 ml	100 ml	100 ml
10X Wash Solution	125 ml	125 ml	125 ml
WestClearTM Nitrocellulose Membrane (0.2 μm, 7.5 X 8 cm)	10 sheets	10 sheets	10 sheets
LumiSensorTM Plus Chemiluminescent HRP Substrate	2 X 30 ml	2 X 30 ml	2 X 30 ml
Protocol	1	1	1

PROTOCOL: 20070102153905 (PDF)

Application Examples:

Comparison of One-Step Western^TM Blot with classical Western Blot using rabbit polyclonal antibody.

One-Step Western^TM Advanced Blot was compared with classical Western Blot for the detection of a His-tagged protein from E. coli cell lysate. Two similar blots were processed with different procedures using Rabbit Anti His-tag (GenScript, A00174): Classical Western Blot detection (4.5 hours, left panel, Figure 2) and One-Step Western^TM Advanced Blot (1.25 hours, right panel, Figure 2).

Figure 2. Western Blots for the detection of a His-tagged protein from E. coli cell lysate by both classical Western and One-Step Western^TM using advanced kit L00241. 200, 80, 32, 12.8 and 5.12 ng of total protein was loaded in Lane 1, 2, 3, 4 and 5, espectively. The classical Western blot was developed with LumiSensor^TM Chemiluminescent HRP Substrate (GenScript, L00221V60) and the One-Step Western^TM blot was developed with LumiSensor^TM Plus Chemiluminescent HRP Substrate included in the kit L00241.

Compared with classical Western blot that takes more than 4.5 hours, One-Step Western^TM Advanced Kit, which takes only 1.25 hours for the detection of protein, produces higher signal with very low background.