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Human Protoporphyrinogen Oxidase / PPOX ELISA Kit

SKU:
HUFI02778
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P50336
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich
Synonyms:
PPOX, Orexin, Hypocretin
Reactivity:
Human
Research Area:
Metabolism
$719
Frequently bought together:

Description

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Human Protoporphyrinogen Oxidase/PPOX ELISA Kit

The Human Protoporphyrinogen oxidase (PPOX) ELISA Kit is a powerful tool for the detection of Protoporphyrinogen oxidase levels in human serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring accurate and reproducible results for a variety of research applications.Protoporphyrinogen oxidase is an enzyme critical for heme biosynthesis and plays a vital role in various physiological processes.

Dysregulation of PPOX has been linked to a range of disorders, including porphyrias and certain types of cancer. By measuring PPOX levels, researchers can gain valuable insights into these conditions and potentially identify new therapeutic targets.Order the Human PPOX ELISA Kit today and take the next step in advancing your research in the field of heme biosynthesis and related disorders.

Product Name:Human Protoporphyrinogen Oxidase / PPOX ELISA Kit
Product Code:HUFI02778
Size:96 Assays
Alias:PPOX, Orexin, Hypocretin
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human PPOX 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 PPOX and the recovery rates were calculated by comparing the measured value to the expected amount of Human PPOX in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)93-10596
EDTA plasma(n=5)85-10292
UFH plasma(n=5)94-10599
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PPOX and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)86-102%90-102%86-103%
EDTA plasma(n=5)82-96%86-97%88-100%
UFH plasma(n=5)82-95%86-100%81-100%
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
UniprotP50336
UniProt Protein Function:PPOX: Catalyzes the 6-electron oxidation of protoporphyrinogen-IX to form protoporphyrin-IX. Defects in PPOX are the cause of variegate porphyria (VP). Porphyrias are inherited defects in the biosynthesis of heme, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors. They are classified as erythropoietic or hepatic, depending on whether the enzyme deficiency occurs in red blood cells or in the liver. PV is the most common form of porphyria in South Africa. It is characterized by skin hyperpigmentation and hypertrichosis, abdominal pain, tachycardia, hypertension and neuromuscular disturbances. High fecal levels of protoporphyrin and coproporphyrin, increased urine uroporphyrins and iron overload are typical markers of the disease. Belongs to the protoporphyrinogen oxidase family.
UniProt Protein Details:Protein type:Cofactor and Vitamin Metabolism - porphyrin and chlorophyll; Oxidoreductase; Mitochondrial; EC 1.3.3.4

Chromosomal Location of Human Ortholog: 1q22

Cellular Component: mitochondrial membrane; mitochondrial intermembrane space; integral to mitochondrial inner membrane; intrinsic to mitochondrial inner membrane

Molecular Function:protoporphyrinogen oxidase activity; FAD binding

Biological Process: response to drug; porphyrin metabolic process; protoporphyrinogen IX biosynthetic process; porphyrin biosynthetic process; heme biosynthetic process

Disease: Porphyria Variegata

NCBI Summary:This gene encodes the penultimate enzyme of heme biosynthesis, which catalyzes the 6-electron oxidation of protoporphyrinogen IX to form protoporphyrin IX. Mutations in this gene cause variegate porphyria, an autosomal dominant disorder of heme metabolism resulting from a deficiency in protoporphyrinogen oxidase, an enzyme located on the inner mitochondrial membrane. Alternatively spliced transcript variants encoding the same protein have been identified. [provided by RefSeq, Jul 2008]
UniProt Code:P50336
NCBI GenInfo Identifier:1709742
NCBI Gene ID:5498
NCBI Accession:P50336.1
UniProt Secondary Accession:P50336,Q5VTW8, D3DVG0,
UniProt Related Accession:P50336
Molecular Weight:477
NCBI Full Name:Protoporphyrinogen oxidase
NCBI Synonym Full Names:protoporphyrinogen oxidase
NCBI Official Symbol:PPOX
NCBI Official Synonym Symbols:VP; PPO; V290M
NCBI Protein Information:protoporphyrinogen oxidase
UniProt Protein Name:Protoporphyrinogen oxidase
UniProt Gene Name:PPOX
UniProt Entry Name:PPOX_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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