GenScript - kit: One-Step Western<sup>TM</sup> Fluorescent Kit

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One-Step Western^TM Fluorescent Kit

Description:

GenScript One-Step Western^TM Fluorescent Kit yields journal-quality Western or Dot blot results in about one hour. Using GenScript breakthrough immunodetection technology (patent pending), the kit replaces the classical three-step Western process, 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 and fluorescent dye labeled secondary antibody for 40 minutes. Then wash three times for ten minutes each. The membrane can then be scanned on a LI-COR Odyssey Infrared Imaging Systems.

The One-Step Western^TM Fluorescent Kit is designed to produce high signal with low background for quick and clear Western analysis of proteins. This kit can be used for any primary antibodies from any species. An appropriate fluorescent dye labeled secondary antibody is also needed.

Key Features

Easy to perform: This kit has fewer and simpler steps.
Reproducible results: The kit produces highly reproducible results.
No dark room, developer or X-Ray film is needed.

Examples:

Fluorescent Western blot detection of GST using rabbit polyclonal antibody
GST protein was detected using One-Step Western^TM Fluorescent Kit (GenScript, L00397). The mixture 1 was prepared by mixing 10 µg of Rabbit Anti-GST-tag Polyclonal Antibody (GenScript, A00097) with 5.0 µg of IR^®800 Goat anti-Rabbit (LI-COR) in 100 µl of WB-1.

Figure 1. Fluorescent Western blots for the detection of GST protein using the One-Step Western^TM Fluorescent Kit (GenScript, L00397). 50.0, 25.0, 12.5, 6.25, 3.12, 1.56, 0.78 and 0.39 ng of GST protein were loaded into lanes 1, 2, 3, 4, 5, 6, 7 and 8 respectively.
Fluorescent Western blot detection of housekeeping protein GAPDH using goat polyclonal antibody
One-Step Western^TM Fluorescent Kit (GenScript, L00397) was used to detect GAPDH protein in HeLa cell lysate. The mixture 1 was prepared by mixing 6 µg of Goat Anti-GAPDH Polyclonal Antibody (GenScript, A00191) with 3 µg of IR^®800 Donkey anti Goat (LI-COR) in 100 µl of WB-1.

Figure 2. Fluorescent Western blots for the detection of GAPDH using the One-Step Western^TM Fluorescent Kit (GenScript, L00397). 5.0, 2.5, 1.25 0.62, 0.31, 0.16, 0.08 and 0.04 µg of HeLa cell lysate were loaded into lanes 1, 2, 3, 4, 5, 6, 7 and 8 respectively.
Protein marker for Fluorescent Western
GenScript also provides protein marker for Fluorescent Western (M00124), the performance of the marker is demonstrated at below.
10 µl and 5 µl of reconstituted protein marker for Fluorescent Western blot were loaded in 12% Express SDS-PAGE Gel (GenScript, MG012W10).
Fig. A shows the results when IRDye 680-conjugated Goat Anti-Rabbit IgG (LI-COR) is used in the Western blot.
Fig. B shows the results when IRDye 800CW-conjugated Goat Anti-Mouse IgG (LI-COR) is used in the Western blot. Images were acquired using Odyssey Infrared Imaging system (LI-COR).

Technical Manual:

Download PDF file TM0353

Components:

code	L00397
Pretreat Solution A	100 ml
Pretreat Solution B	100 ml
WB-1 Solution	2 ml
WB-2 Solution	100 ml
10X Wash Solution	125 ml
Protocol	1

One-Step WesternTM Fluorescent Kit