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Human CYP24A1 ELISA Kit

SKU:
HUFI02374
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q07973
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich
Synonyms:
CYP24A1, CL1, CLICK1, DCAMKL1, DCDC3A, DCLK
Reactivity:
Human
Research Area:
Cell Biology
$719
Frequently bought together:

Description

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Human CYP24A1 ELISA Kit

The Human CYP24A1 ELISA Kit is specifically designed for the quantitative detection of CYP24A1 levels in human serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, providing accurate and consistent results for various research applications.CYP24A1 is an enzyme crucial for the regulation of vitamin D metabolism, involved in the breakdown of active vitamin D metabolites. Dysregulation of CYP24A1 has been linked to various diseases, including vitamin D-related disorders, cancer, and autoimmune conditions.

Thus, this ELISA kit serves as an essential tool for studying the role of CYP24A1 in these diseases and potential therapeutic interventions.Overall, the Human CYP24A1 ELISA Kit is a reliable and efficient solution for researchers looking to investigate the role of CYP24A1 in health and disease.

Product Name:Human CYP24A1 ELISA Kit
Product Code:HUFI02374
Size:96 Assays
Alias:CYP24A1, CL1, CLICK1, DCAMKL1, DCDC3A, DCLK
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human CYP24A1 concentrations in serum plasma and other biological fluids.
Sensitivity:0.094ng/ml
Range:0.156-10ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human CYP24A1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CYP24A1 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)90-10296
EDTA plasma(n=5)89-10598
UFH plasma(n=5)88-9995
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CYP24A1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-101%86-105%94-100%
EDTA plasma(n=5)82-100%83-98%87-101%
UFH plasma(n=5)82-100%80-94%81-91%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment 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
UniprotQ07973
UniProt Protein Function:CYP24A1: Has a role in maintaining calcium homeostasis. Catalyzes the NADPH-dependent 24-hydroxylation of calcidiol (25- hydroxyvitamin D(3)) and calcitriol (1-alpha,25-dihydroxyvitamin D(3)). The enzyme can perform up to 6 rounds of hydroxylation of calcitriol leading to calcitroic acid. It also shows 23- hydroxylating activity leading to 1-alpha,25-dihydroxyvitamin D(3)-26,23-lactone as end product. Defects in CYP24A1 are the cause of hypercalcemia infantile (HCAI). HCAI is a disorder characterized by abnormally high level of calcium in the blood, failure to thrive, vomiting, dehydration, and nephrocalcinosis. Belongs to the cytochrome P450 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:Protein type:EC 1.14.13.126; Mitochondrial; Oxidoreductase

Chromosomal Location of Human Ortholog: 20q13

Cellular Component: mitochondrial inner membrane

Molecular Function:iron ion binding; heme binding; 25-hydroxycholecalciferol-24-hydroxylase activity; oxidoreductase activity; 1-alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) 24-hydroxylase activity

Biological Process: osteoblast differentiation; steroid metabolic process; response to vitamin D; vitamin metabolic process; xenobiotic metabolic process; vitamin D metabolic process; vitamin D catabolic process

Disease: Hypercalcemia, Infantile

NCBI Summary:This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This mitochondrial protein initiates the degradation of 1,25-dihydroxyvitamin D3, the physiologically active form of vitamin D3, by hydroxylation of the side chain. In regulating the level of vitamin D3, this enzyme plays a role in calcium homeostasis and the vitamin D endocrine system. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:Q07973
NCBI GenInfo Identifier:19862747
NCBI Gene ID:1591
NCBI Accession:Q07973.2
UniProt Secondary Accession:Q07973,Q15807, Q32ML3, Q5I2W7,
UniProt Related Accession:Q07973
Molecular Weight:59kDa
NCBI Full Name:1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial
NCBI Synonym Full Names:cytochrome P450, family 24, subfamily A, polypeptide 1
NCBI Official Symbol:CYP24A1
NCBI Official Synonym Symbols:CP24; HCAI; CYP24; P450-CC24
NCBI Protein Information:1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial; 1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial; 24-OHase; cytochrome P450 24A1; cytochrome P450-CC24; vitamin D 24-hydroxylase; exo-mitochondrial protein; vitamin D(3) 24-hydroxylase; 1,25-@dihydroxyvitamin D3 24-hydroxylase; cytochrome P450, subfamily XXIV (vitamin D 24-hydroxylase)
UniProt Protein Name:1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial
UniProt Synonym Protein Names:Cytochrome P450 24A1; Cytochrome P450-CC24
Protein Family:1,25-dihydroxyvitamin D(3) 24-hydroxylase
UniProt Gene Name:CYP24A1
UniProt Entry Name:CP24A_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct 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.

StepProtocol
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!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
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.
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.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
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.
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.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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.

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