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Human CYP7B1(Cytochrome P450 7B1) ELISA Kit (HUFI03196)

SKU:
HUFI03196
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O75881
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich
Synonyms:
Cytochrome P450 7B1
Reactivity:
Human
€599

Description

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Human CYP7B1 (Cytochrome P450 7B1) ELISA Kit

The Human CYP7B1 (Cytochrome P450 7B1) ELISA Kit is a highly sensitive and specific assay designed for the accurate detection of CYP7B1 levels in human serum, plasma, and cell culture supernatants. This kit provides reliable and reproducible results, making it ideal for various research applications.CYP7B1 is a key enzyme in the metabolism of cholesterol and bile acid synthesis, playing a crucial role in maintaining lipid homeostasis. Dysregulation of CYP7B1 has been implicated in various metabolic disorders, including cardiovascular diseases, liver diseases, and hormonal imbalances.

Therefore, measuring CYP7B1 levels can provide valuable insights into these conditions and aid in the development of potential therapeutic strategies.Overall, the Human CYP7B1 ELISA Kit offers researchers a powerful tool for studying the role of CYP7B1 in health and disease, ultimately contributing to the advancement of knowledge and healthcare interventions in this important area of research.

Product Name:Human CYP7B1(Cytochrome P450 7B1) ELISA Kit
Product Code:HUFI03196
Size:96 Assays
Alias:Cytochrome P450 7B1
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human CYP7B1 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 CYP7B1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CYP7B1 in samples. Enquire for more information.
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CYP7B1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
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
UniprotO75881
UniProt Protein Function:CYP7B1: Defects in CYP7B1 are the cause of spastic paraplegia autosomal recessive type 5A (SPG5A). Spastic paraplegia is a neurodegenerative disorder characterized by a slow, gradual, progressive weakness and spasticity of the lower limbs. Rate of progression and the severity of symptoms are quite variable. Initial symptoms may include difficulty with balance, weakness and stiffness in the legs, muscle spasms, and dragging the toes when walking. In some forms of the disorder, bladder symptoms (such as incontinence) may appear, or the weakness and stiffness may spread to other parts of the body. Defects in CYP7B1 are the cause of congenital bile acid synthesis defect type 3 (CBAS3). Clinical features include severe cholestasis, cirrhosis and liver synthetic failure. Hepatic microsomal oxysterol 7-alpha-hydroxylase activity is undetectable. Belongs to the cytochrome P450 family.
UniProt Protein Details:Protein type:EC 1.14.13.100; Oxidoreductase; Endoplasmic reticulum; Lipid Metabolism - primary bile acid biosynthesis

Chromosomal Location of Human Ortholog: 8q21.3

Cellular Component: endoplasmic reticulum membrane

Molecular Function:oxysterol 7-alpha-hydroxylase activity

Biological Process: bile acid biosynthetic process; sterol metabolic process

Disease: Bile Acid Synthesis Defect, Congenital, 3; Spastic Paraplegia 5a, Autosomal Recessive

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 endoplasmic reticulum membrane protein catalyzes the first reaction in the cholesterol catabolic pathway of extrahepatic tissues, which converts cholesterol to bile acids. This enzyme likely plays a minor role in total bile acid synthesis, but may also be involved in the development of atherosclerosis, neurosteroid metabolism and sex hormone synthesis. Mutations in this gene have been associated with hereditary spastic paraplegia (SPG5 or HSP), an autosomal recessive disorder. [provided by RefSeq, Apr 2016]
UniProt Code:O75881
NCBI GenInfo Identifier:20141320
NCBI Gene ID:9420
NCBI Accession:O75881.2
UniProt Secondary Accession:O75881,Q9UNF5, B2RN07,
UniProt Related Accession:O75881
Molecular Weight:58,256 Da
NCBI Full Name:25-hydroxycholesterol 7-alpha-hydroxylase
NCBI Synonym Full Names:cytochrome P450 family 7 subfamily B member 1
NCBI Official Symbol:CYP7B1  
NCBI Official Synonym Symbols:CP7B; CBAS3; SPG5A  
NCBI Protein Information:25-hydroxycholesterol 7-alpha-hydroxylase
UniProt Protein Name:25-hydroxycholesterol 7-alpha-hydroxylase
UniProt Synonym Protein Names:Cytochrome P450 7B1; Oxysterol 7-alpha-hydroxylase
Protein Family:25-hydroxycholesterol 7-alpha-hydroxylase
UniProt Gene Name:CYP7B1  
UniProt Entry Name:CP7B1_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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