The Mouse Sphingosine Kinase 1 (SPHK1) ELISA Kit is a reliable tool for accurately measuring levels of SPHK1 in mouse serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit ensures consistent and dependable results for a variety of research applications.SPHK1 is an important enzyme that plays a key role in sphingolipid metabolism and signaling pathways. It is involved in regulating cell proliferation, apoptosis, migration, and inflammation, making it a valuable target for studying various diseases and developing potential therapies.
By using the Mouse SPHK1 ELISA Kit, researchers can gain valuable insights into the role of SPHK1 in different biological processes and disease mechanisms, ultimately advancing our understanding of complex pathways and potential treatment options.
Product Name:
Mouse Sphingosine kinase 1 (Sphk1) ELISA Kit
SKU:
MOEB1971
Size:
96T
Target:
Mouse Sphingosine kinase 1 (Sphk1)
Synonyms:
Acetyltransferase SPHK1, SK 1, Sk1
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Mouse
Detection Range:
31.2-2000pg/ml
Sensitivity:
15.75pg/mL
Intra CV:
5.9%
Inter CV:
8.3%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
102-114%
89-99%
102-114%
100-110%
EDTA Plasma(N=5)
102-114%
105-115%
81-90%
103-113%
Heparin Plasma(N=5)
96-105%
106-116%
108-119%
101-111%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
106
100-112
Plasma
108
102-114
Function:
Has serine acetyltransferase activity on PTGS2/COX2 in an acetyl-CoA dependent manner. The acetyltransferase activity increases in presence of the kinase substrate, sphingosine. During neuroinflammation, through PTGS2 acetylation, promotes neuronal secretion of specialized preresolving mediators (SPMs), especially 15-R-lipoxin A4, which results in an increase of phagocytic microglia.
Uniprot:
Q8CI15
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Sphingosine kinase 1
Sub Unit:
Interacts with ACY1 (PubMed:15196915). Binds to calmodulin. Interacts with SPHKAP (By similarity). Interacts with CIB1, the interaction occurs in a calcium-dependent manner (By similarity). Interacts with TRAF2 (By similarity). Interacts with EEF1A1; the interaction enhances SPHK1 kinase activity (By similarity).
Research Area:
Cardiovascular
Subcellular Location:
Cytoplasm Endosome membrane Peripheral membrane protein Nucleus Cell membrane Cell junction Synapse Translocated from the cytoplasm to the plasma membrane in a CIB1-dependent manner. Binds to membranes containing negatively charged lipids but not neutral lipids (By similarity). Recruited to endocytic membranes by sphingosine where promotes membrane fusion (PubMed:27806293).
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
SPHK1: Catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (SPP), a lipid mediator with both intra- and extracellular functions. Also acts on D-erythro-sphingosine and to a lesser extent sphinganine, but not other lipids, such as D,L-threo-dihydrosphingosine, N,N-dimethylsphingosine, diacylglycerol, ceramide, or phosphatidylinositol. Interacts with ACY1. Binds to calmodulin. Interacts with SPHKAP. Widely expressed with highest levels in adult liver, kidney, heart and skeletal muscle. 2 isoforms of the human protein are produced by alternative splicing.Protein type: Lipid Metabolism - sphingolipid; EC 2.7.1.91; Kinase, lipidCellular Component: synaptic vesicle; neuron projection; membrane; axon; cytoplasm; plasma membrane; nucleus; cytosolMolecular Function: D-erythro-sphingosine kinase activity; transferase activity; calmodulin binding; DNA binding; protein phosphatase 2A binding; sphinganine kinase activity; magnesium ion binding; nucleotide binding; kinase activity; diacylglycerol kinase activity; ATP binding; NAD+ kinase activityBiological Process: blood vessel development; metabolic process; cyclooxygenase pathway; calcium-mediated signaling; positive regulation of NF-kappaB import into nucleus; sphingosine metabolic process; positive regulation of mitotic cell cycle; lipid phosphorylation; positive regulation of cell growth; positive regulation of neurotransmitter secretion; activation of NF-kappaB transcription factor; positive regulation of angiogenesis; positive regulation of fibroblast proliferation; positive regulation of protein ubiquitination; regulation of interleukin-1 beta production; positive regulation of cell proliferation; protein kinase C activation; positive regulation of protein amino acid phosphorylation; positive regulation of smooth muscle contraction; brain development; inflammatory response; phosphorylation; negative regulation of apoptosis; positive regulation of cell migration
UniProt Protein Details:
NCBI Summary:
This gene encodes a kinase that phosphorylates sphingosine into sphingosine-1-phosphate, which is involved in cell differentiation, motility, and apoptosis. The encoded protein plays a role in maintaining cellular levels of sphingosine-1-phosphate. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Feb 2010]
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
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.
