ONE-HOUR IP-Western Kit (Rabbit) (5 assays/kit)

ONE-HOUR IP-Western Kit (Rabbit)

Description

GenScript ONE-HOUR IP-Western Kit contains all the necessary reagents, buffers, nitrocellulose membrane and HRP substrate for performing an IP-Western blot. It was developed using breakthrough immuno-detection technology (patent pending), which allows the user to perform a complete IP-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 five minutes and follow with incubation in WB solution (prepared from primary antibody, IP-WB 1, IP-WB 2 and IP-WB 3) for 40 minutes. Wash the membrane three times for five minutes each and then the blot can be developed using the LumiSensor^TM Chemiluminescent HRP Substrate Kit included in the kit.

The ONE-HOUR IP-Western Kit (L00231) with rabbit antibody allows for the detection of nanograms of antigen while leaving quantities of rabbit primary antibody (or any other antibody) of up to 2 µg per lane undetected, giving a clearer, more useful result. With this kit, protein G blocker is only needed if protein G or A/G resin is used during immunoprecipitation. Protein A carryover does not affect the Western results.

The results of the ONE-HOUR IP-Western blot result are compared to results of a conventional three-step Western in figure 1 below.
 

Key Features

• Easy to perform: Fewer steps mean fewer chances for human error.
• Compatible with immunoprecipitation methods incorporating protein A, G, or A/G resin or any secondary antibody.
• Low background: The kit contains WestClear^TM Nitrocellulose Membrane and LumiSensor^TM 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.
• Needs far less optimization than the classical three-step method.
• No secondary antibody is needed.

Applications

The ONE-HOUR IP-Western Kit enables fast Western and Dot blots for the detection of proteins from immunoprecipitation. It is compatible with the following resins:

• Protein A, G, or A/G resin
• Any secondary antibody resin

Examples

Western blot detection using mouse primary antibody.
Western blots for the detection of multiple tag fusion protein using Rabbit Anti-GST-tag Polyclonal Antibody (GenScript, A00097) by both classical Western and ONE-HOUR IP-Western (using kit L00231). Both blots were developed with the LumiSensor^TM Chemiluminescent HRP Substrate included in kit L00231.

Figure 1. Western blot detection of GST protein by both classical western and ONE-HOUR IP-Western (using kit L00231). Both blots are developed using the LumiSensor^TM Chemiluminescent HRP Substrate included in kit L00231.

Technical Manual

Components