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Human S1P (Sphingosine 1 Phosphate) ELISA Kit (HUFI04787)

SKU:
HUFI04787
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q14703
Sensitivity:
1.875ng/ml
Range:
3.125-200ng/ml
ELISA Type:
Competitive
Synonyms:
S1P
Reactivity:
Human
$719
Frequently bought together:

Description

Human S1P (Sphingosine 1 Phosphate) ELISA

S1P (Sphingosine 1 Phosphate) encodes a type 1 membrane bound protease which is ubiquitously expressed and regulates cholesterol or lipid homeostasis via cleavage of substrates at non-basic residues. S1P (Sphingosine 1 Phosphate) undergoes autocatalytic processing events in the ER and cis/medial-Golgi. Mutations in S1P (Sphingosine 1 Phosphate) may be associated with lysosomal dysfunction. Diseases associated with S1P (Sphingosine 1 Phosphate) include Spondyloepiphyseal Dysplasia and Hemorrhagic Fever.

system_update_alt Datasheet system_update_alt MSDS

Key Features

Save Time Pre-coated 96 well plate
Quick Start Kit includes all necessary reagents
Publication Ready Reproducible and reliable results

Overview

Product Name:

Human S1P (Sphingosine 1 Phosphate) ELISA Kit

Product Code:

HUFI04787

Size:

96 Assays

Alias:

S1P ELISA Kit

Detection Method:

Competitive ELISA, Coated with Antibody

Application:

This immunoassay kit allows for the in vitro quantitative determination of Human S1P (Sphingosine 1 Phosphate) concentrations in serum plasma and other biological fluids.

Sensitivity:

< 1.875ng/ml

Range:

3.125-200ng/ml

Storage:

4°C for 6 months

Note:

For Research Use Only

Additional Information

Recovery

Matrices listed below were spiked with certain level of Human S1P (Sphingosine 1 Phosphate) and the recovery rates were calculated by comparing the measured value to the expected amount of Human S1P (Sphingosine 1 Phosphate) in samples.

Matrix

Recovery Range (%)

Average (%)

serum(n=5)

87-105

98

EDTA plasma(n=5)

85-102

91

heparin plasma(n=5)

85-98

91

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human S1P (Sphingosine 1 Phosphate) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample

1:2

1:4

1:8

serum(n=5)

87-103%

89-95%

93-102%

EDTA plasma(n=5)

83-98%

85-98%

86-99%

UFH plasma(n=5)

86-100%

81-99%

84-100%

CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Kit Components

Component Quantity Storage

ELISA Microplate (Dismountable)

8x12 strips

4°C for 6 months

Lyophilized Standard

2

4°C/ -20°C

Sample/Standard Dlution Buffer

20ml

4°C

Biotin-labeled Antibody (Concentrated)

120ul

4°C (Protection from light)

Antibody Dilution Buffer

10ml

4°C

HRP-Streptavidin Conjugate (SABC)

120ul

4°C (Protect from light)

SABC Dilution Buffer

10ml

4°C

TMB Substrate

10ml

4°C (Protection from light)

Stop Solution

10ml

4°C

Wash Buffer (25X)

30ml

4°C

Plate Sealer

5

-

Other materials required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

Protein Information

Heading 1 Heading 2

UniProt

NCBI Gene ID

NCBI GenInfo Identifier

NCBI Full Name

Membrane-bound transcription factor site-1 protease

Protein Family

Membrane-bound transcription factor site-1 protease

Protocol

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Equilibrate the TMB substrate for at least 30 min at 37°C beforeuse. When diluting samples and reagents, they must be mixed completely andevenly. It is recommended to plot a standard curve for each test.

Step Procedure

1.

Set standard, test sample and control (zero) wells on the pre-coatedplate respectively, and then, record their positions. It isrecommended to measure each standard and sample in duplicate. Washplate 2 times before adding standard, sample and control (zero) wells!

2.

Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blankwell is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody workingsolution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thoroughmixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).

3.

Wash: Aspirate each well and wash, repeating the process three timesWash by filling each well with Wash Buffer (approximately 350µL)using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essentialto good performance. After the last wash, remove any remaining WashBuffer by aspirating or decanting. Invert the plate and pat it againstthick clean absorbent paper.

4.

HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC workingsolution to each well. Cover with a new Plate sealer. Incubate for30minutes at 37°C.

5.

Wash: Repeat the aspiration/wash process for five times.

6.

TMB Substrate: Add 90µL of TMB Substrate to each well. Coverwith a new Plate sealer. Incubate for about 10-20 minutes at 37°C.Protect from light. The reaction time can be shortened or extendedaccording to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, you can terminatethe reaction.

7.

Stop: Add 50µL of Stop Solution to each well. Color turn toyellow immediately. The adding order of stop solution should be as thesame as the substrate solution.

8.

OD Measurement: Determine the optical density (OD Value) of each wellat once, using a microplate reader set to 450 nm. You should open themicroplate reader ahead, preheat the instrument, and set the testing parameters.

Sample Type

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Tissue homogenates

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C

Breast Milk

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

S1P Background

S1P Background

Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule that serves as a critical signaling mediator in numerous biological processes. It is synthesized through the metabolism of sphingolipids and acts as a potent regulator of cell behavior. S1P exerts its effects by binding to s coupled receptors, known as S1P receptors, which are widely expressed in various cell types. Through its interactions with these receptors, S1P modulates cellular processes such as cell growth, survival, migration, and immune responses. Additionally, S1P has been implicated in the regulation of vascular integrity, lymphocyte trafficking, and neuroinflammation. The dysregulation of S1P signaling has been linked to several diseases, including cancer, autoimmune disorders, and cardiovascular conditions.

S1P Receptors

Sphingosine-1-phosphate (S1P) is released from cells into the extracellular environment via specific transporters, such as spinster homolog 2 (Spns2) or ABC transporters. Once in the extracellular milieu, S1P binds to a family of plasma membrane G protein-coupled receptors known as S1P receptors (S1P1–S1P5), initiating a range of biological responses. Simultaneously, intracellularly generated S1P, facilitated by the enzyme SK2, interacts with various targets, including HDAC-1/2, human telomerase reverse transcriptase (hTERT), and prohibitin 2. As a result, the activity of SK2 plays a significant role in diverse and crucial biological events, such as epigenetic regulation, aging, and the assembly of mitochondrial respiratory complexes.

S1P ELISA Kit FAQsS

What is the S1P ELISA Kit used for?

By detecting and quantifying S1P levels, the S1P ELISA kit provides researchers with a valuable tool to investigate the role of S1P in various biological processes and disease pathways, facilitating a deeper understanding of its mechanisms and potential therapeutic applications.

What are the advantages of using the S1P ELISA Kit?

The S1P ELISA Kit offers several advantages, including high sensitivity, accuracy, and reproducibility. It provides a user-friendly and reliable method to quantify S1P levels in biological specimens, allowing for precise measurements and robust data analysis.

What sample types are compatible with S1P ELISA Kit?

The S1P ELISA Kit is compatible with various sample types, including serum, plasma, cell lysates, and tissue homogenates. It provides flexibility in sample selection, allowing researchers to analyze S1P levels in different biological matrices.

What are the storage requirements with S1P ELISA Kit?

The S1P ELISA Kit components should be stored according to the instructions provided in the kit manual. Generally, it is recommended to store the kit components at the recommended temperature to ensure their stability and optimal performance.

What should I do if my assay results are not optimal?

If you encounter any issues or have suboptimal assay results, we recommend contacting our dedicated support team for assistance. They will be available to provide troubleshooting guidance, answer your questions, and ensure you achieve the best possible results with the S1P ELISA Kit.