Human Ceramide kinase / CERK ELISA Kit
- SKU:
- HUFI01256
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- Q8TCT0
- Sensitivity:
- 0.375ng/ml
- Range:
- 0.625-40ng/ml
- ELISA Type:
- Sandwich
- Synonyms:
- CERK, Ceramide kinase, hCERK, LK4, Acylsphingosine kinase, Lipid kinase 4
- Reactivity:
- Human
- Research Area:
- Cell Biology
Description
Human Ceramide kinase / CERK ELISA Kit
Ceramide is activated to ceramide 1-phosphate (C1P), a sphingolipid metabolite, by Ceramide kinase/CERK. Both Ceramide kinase/CERK and C1P have been linked to various cell functions, including proliferation, apoptosis, phagocytosis, and inflammation. Ceramide kinase/CERK is associated with a number of diseases, including Retinitis Pigmentosa 26 and Cochlear Disease. Downstream effects associated with Ceramide kinase/CERK include the Ceramide Pathway and Sphingolipid Metabolism.
Key Features
| Save Time | Pre-coated 96 well plate | |
| Quick Start | Kit includes all necessary reagents | |
| Publication Ready | Reproducible and reliable results |
Overview
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Product Name: |
Human Ceramide kinase / CERK ELISA Kit |
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Product Code: |
HUFI01256 |
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Size: |
96 Assays |
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Alias: |
CERK, Ceramide kinase, hCERK, LK4, Acylsphingosine kinase, Lipid kinase 4 |
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Detection Method: |
Sandwich ELISA, Double Antibody |
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Application: |
This immunoassay kit allows for the in vitro quantitative determination of Human CERK concentrations in serum plasma and other biological fluids. |
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Sensitivity: |
0.375ng/ml |
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Range: |
0.625-40ng/ml |
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Storage: |
4°C for 6 months |
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Note: |
For Research Use Only |
Additional Information
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Recovery: |
Matrices listed below were spiked with certain level of Human CERK and the recovery rates were calculated by comparing the measured value to the expected amount of Human CERK in samples.
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Linearity: |
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CERK and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
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CV(%): |
Intra-Assay: CV<8% |
Kit Components
| Component | Quantity | Storage |
|
ELISA Microplate (Dismountable) |
8x12 strips |
4°C for 6 months |
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Lyophilized Standard |
2 |
4°C/ -20°C |
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Sample/Standard Dlution Buffer |
20ml |
4°C |
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Biotin-labeled Antibody (Concentrated) |
120ul |
4°C (Protection from light) |
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Antibody Dilution Buffer |
10ml |
4°C |
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HRP-Streptavidin Conjugate (SABC) |
120ul |
4°C (Protect from light) |
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SABC Dilution Buffer |
10ml |
4°C |
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TMB Substrate |
10ml |
4°C (Protection from light) |
|
Stop Solution |
10ml |
4°C |
|
Wash Buffer (25X) |
30ml |
4°C |
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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
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Uniprot: |
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UniProt Protein Function: |
CERK: Catalyzes specifically the phosphorylation of ceramide to form ceramide 1-phosphate. Acts efficiently on natural and analog ceramides (C6, C8, C16 ceramides, and C8-dihydroceramide), to a lesser extent on C2-ceramide and C6-dihydroceramide, but not on other lipids, such as various sphingosines. Binds phosphoinositides. |
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UniProt Code: |
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NCBI GenInfo Identifier: |
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NCBI Gene ID: |
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NCBI Accession: |
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UniProt Related Accession: |
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Molecular Weight: |
60kDa |
Protocol
*Note: Protocols are specific to each batch/lot. For the exact instructions please follow the protocol included in your kit.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. 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-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells! |
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2. |
Aliquot 0.1ml standard solutions into the standard wells. |
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3. |
Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well. |
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4. |
Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells. |
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5. |
Seal the plate with a cover and incubate at 37 °C for 90 min. |
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6. |
Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2. |
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7. |
Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall. |
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8. |
Seal the plate with a cover and incubate at 37°C for 60 min. |
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9. |
Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash. |
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10. |
Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min. |
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11. |
Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min. |
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12. |
Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color. |
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13. |
Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. |
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14. |
Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. |
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 |
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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. |
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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. |
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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. |
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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. |
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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. |
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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. |
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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 |
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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. |
CERK Background
Ceramide kinase, also known as CERK (Ceramide Kinase), is an enzyme involved in the metabolism of ceramide, a type of lipid molecule found in cell membranes. Ceramide is an important component of cellular signaling pathways and plays a role in various cellular processes, including cell growth, differentiation, apoptosis, and inflammation.
Function of CERK
CERK is responsible for converting ceramide into ceramide-1-phosphate (C1P) by phosphorylating ceramide using ATP as a phosphate donor. This enzymatic reaction adds a phosphate group to the ceramide molecule, resulting in the formation of ceramide-1-phosphate.
Conversion of Ceramide to Ceramide-1-phosphate (C1P)
Ceramide-1-phosphate has been identified as a bioactive lipid molecule that regulates several cellular functions. It acts as a signaling molecule involved in the modulation of cell survival, proliferation, and migration. It also plays a role in immune responses and inflammatory processes.
CERK FAQs
What is the CERK ELISA Kit?
The Human Ceramide Kinase / CERK ELISA Kit is a specialized kit designed to measure the levels of CERK in human samples.
What are the advantages of using the CERK ELISA Kit?
The Human Ceramide Kinase / CERK ELISA Kit offers several advantages. It provides accurate quantification of CERK levels in human samples, enabling precise assessment of CERK activity or expression.
Where can I find more information about the CERK ELISA Kit?
For more detailed information about the CERK ELISA Kit, including technical specifications, performance characteristics, and ordering details, please refer to the product brochure or contact our customer support team. We are here to assist you with any inquiries you may have.
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