Rat Sphingosine -1-phosphate, S1P ELISA Kit

Catalog No.: BZEK2364

Size: 96T

Range: 50 nmol/L - 800 nmol/L

Sensitivity: 5 nmol/L

Storage and Expiration: Store at 2-8℃ for 6 months. 

Application: For quantitative detection of S1P in rat serum, plasma, tissue homogenate or cell culture supernatant.

Introduction

Sphingosine-1-phosphate (S1P) is a signaling sphingolipid, also known as lysosphingolipid. It is also referred to as a bioactive lipid mediator. Sphingolipids at large form a class of lipids characterized by a particular aliphatic aminoalcohol, which is sphingosine. S1P is formed from ceramide, which is composed of a sphingosine and a fatty acid. Ceramidase, an enzyme primarily present in plasma membrane, will convert ceramide to sphingosine. sphingosine is then phosphorylated by sphingosine kinase (SK) isoenzymes. There are two identified isoenzymes, SK1 and SK2. These two enzymes have different tissue distribution. SK1 is highly expressed in spleen, lung and leukocytes, while SK2 is highly expressed in liver and kidney. SK2 is located mainly in the mitochondria, nucleus and the endoplasmic reticulum whereas SK1 is mainly located in cytoplasm and the cell membrane.

Principle of the Assay  

This kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. The purified anti- S1P antibody was pre-coated onto 96-well plates. And the HRP conjugated anti- S1P antibody was used as detection antibodies. The standards, test samples and HRP conjugated detection antibody were added to the wells subsequently, mixed and incubated, then, unbound conjugates were washed away with wash buffer. TMB substrates (A & B) were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the S1P amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then the concentration of S1P can be calculated.

Kit components

1. One 96-well plate pre-coated with anti- rat S1P antibody

2. Standard: 0.3 ml × 6 tubes (0 nmol/L, 50 nmol/L, 100 nmol/L, 200 nmol/L, 400 nmol/L, 800 nmol/L)

3. Wash buffer (20×): 25 ml. Dilution: 1:20

4. Sample diluent buffer: 6 ml

5. HRP conjugated anti- rat S1P antibody (RTU): 10 ml

6. Stop solution: 6 ml

7. TMB substrate A:  6ml

8. TMB substrate B:  6ml

9. Plate sealer:   2

10. Hermetic bag:  1

Material Required But Not Provided

1. 37℃ incubator
2. Microplate reader (wavelength: 450nm)
3. Precise pipette and disposable pipette tips
4. Automated plate washer
5. ELISA shaker
6. 1.5ml of Eppendorf tubes
7. Absorbent filter papers
8. Plastic or glass container with volume of above 1L