Description

Can Get Signal™ is a solution containing an accelerator for antigen-antibody reactions. This reagent improves sensitivity, specificity, and signal-to-noise ration (S/N) for Western blotting, dot blotting, enzyme-linked immunosorbent assay (ELISA), etc. Solutions 1 and 2 refer to the reactions of the primary and secondary antibodies, respectively.

Features

• Enhances immunoassay signals up to several dozen times by maintaining low background signals.
• Can be used in combination with secondary antibodies labeled with peroxidase or alkaline phosphatase, etc.
• Can be used directly without dilution (ready-to-use).

Application

・Western blotting, dot blotting
・Enzyme-linked immunosorbent assay (ELISA),

Specification

Category	Item	Details
Storage condition	-	Store at 4ºC

Components

This kit includes the following components. All reagents should be stored at 4ºC, and protected from light.

Reagent Name	Code No.
Reagent Name	NKB-101T	NKB-101	NKB-201	NKB-301
Solution 1 for primary antibody	50 mL	250 mL	250 mL	-
Solution 2 for secondary antibody	50 mL	250 mL	-	250 mL

Typical reaction flow

Flow chart of western blotting with Can Get Signal™

Flow chart of ELISA with Can Get Signal™

Application Data

Example 1.Detection of phosphorylated proteins by Western blotting

Phosphorylated Akt and ERK were detected by Western blotting analysis using Can Get Signal™ and a conventional method (TBS-T). As a result, the signal intensities of the target bands obtained with Can Get Signal™ were greater than those of the conventional method. The background level of the experiment with Can Get Signal™ was also significantly lower than that of the conventional method. The results suggest that Can Get Signal™ improves the sensitivity and specificity of Western blotting analysis.

Fig. 1. Detection of phosphorylated protein kinases (p-Akt, p-ERK1 and p-ERK2) by Western blotting with Can Get Signal™ and a conventional method
Sample:Cultured bovine adrenal medulla cells
1. Control (H2O)
2. Insulin (1 nM, stimulated for 5 min)
3. Insulin (10 nM, stimulated for 5 min)
4. Insulin (100 nM, stimulated for 5 min)

Antibodies:
<p-Akt>
Primary antibody:Anti Phospho-Akt rabbit polyclonal antibody (1:2,000 dilutioin) Secondary antibody:Anti rabbit-HRP antibody (1:20,000 dilution)
<p-ERK>
Primary antibody:Anti Phospho-ERK monoclonal antibody (1:2,000 dilutioin) Secondary antibody:Anti mouse-HRP antibody (1:20,000 dilution)

*The data was kindly provided by Dr. Yanagita from the Department of Pharmacology, Faculty of Medicine, University of Miyazaki.

Example 2. Detection of His-tagged proteins by Western blotting

His-tagged recombinant proteins were detected with Can Get Signal™ and a conventional method (TBS-T). Can Get Signal™ showed excellent greater sensitivity than the conventional method.

Fig. 2. Detection of His-tagged proteins by Western blotting
1: 6xHis-Catalase fusion protein
2: 6xHis-Catalase fusion protein
3: 6xHis-c-fos
4: 6xHis-c-jun

Antibodies:
Primary antibody:Anti his-tag rabbit polyclonal antibody (1:2,000 dilutioin) Secondary antibody:Anti rabbit-IgG-HRP antibody (1:20,000 dilution)

Example 3. Detection of His-tagged proteins by ELISA

Sandwich ELISA (solid phase antibody: anti-ERK2 monoclonal antibody, primary antibody: anti-His tag polyclonal antibody, secondary antibody: anti-rabbit IgG-HRP antibody) was performed to detect his-tagged human MAP kinase (His-ERK2) synthesized by a cell-free protein synthesis system. Can Get Signal™ showed an excellent quantitative curve as a function of antigen concentration whereas the conventional method with TBS-T resulted in low signals.

Fig. 3. Detection of His-tagged proteins by sandwich ELISA

Documents

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Can Get Signal Immunoreaction Enhancer Solution-Toyobo