Rat Aryl hydrocarbon Receptor/AHR ELISA Kit

Product Name:	Rat AhR (Aryl Hydrocarbon Receptor) ELISA Kit
Product Code:	RTFI00562
Size:	96 Assays
Target:	Rat AhR
Alias:	Ahr, Aryl hydrocarbon receptor, BHLHE76, Ah receptor, AH-receptor, aromatic hydrocarbon receptor, Class E basic helix-loop-helix protein 76
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat AhR and the recovery rates were calculated by comparing the measured value to the expected amount of Rat AhR in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-104	94
EDTA plasma(n=5)	86-100	94
UFH plasma(n=5)	87-99	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat AhR and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	88-103%	86-97%	85-103%
EDTA plasma(n=5)	85-99%	89-99%	82-101%
UFH plasma(n=5)	86-95%	80-99%	81-100%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P41738

UniProt Protein Function:	AHR: a nuclear receptor for aryl hydrocarbons involved in xenobiotic metabolism, cell cycle regulation, and development in response to both endogenous and environmental signals. AhR was initially identified as a receptor for dioxins, which are environmental pollutants generated by waste incineration and other industrial processes. AhR ligands include polycyclic aromatic hydrocarbons, including the carcinogen benzo(a)pyrene and other components of cigarette smoke. Naturally occurring AhR ligands include flavonoids, which are aromatic plant secondary compounds commonly found in vegetables and fruits. Cytoplasmic aryl hydrocarbon receptors are found in protein complexes with heat shock proteins. Upon ligand binding, AhR dissociates from heat shock proteins and translocate to the nucleus where it dimerizes with AhR nuclear translocator (ARNT, HIF-1b). The AhR/ARNT heterodimer binds to nuclear xenobiotic response elements to control the expression of genes associated with xenobiotic metabolism, including several cytochrome P450 genes. AhR is ubiquitously expressed and is thought to play a role in regulation of cell proliferation and differentiation, cytokine expression, and xenobiotic metabolism. Research studies link AhR activity with the control of regulatory T-cell and T-helper 17 cell differentiation, regulation of the inflammatory response, and the onset of lung cancer.
UniProt Protein Details:	Protein type:Transcription factor; Nuclear receptor; DNA-binding Cellular Component: cytoplasm; cytosol; nucleoplasm; nucleus Molecular Function:aryl hydrocarbon receptor activity; DNA binding; Hsp90 protein binding; ligand-dependent nuclear receptor activity; protein binding; protein dimerization activity; protein heterodimerization activity; RNA polymerase II transcription factor activity, enhancer binding; sequence-specific DNA binding; transcription factor activity; transcription factor binding Biological Process: apoptosis; B cell homeostasis; blood circulation; blood vessel development; blood vessel morphogenesis; blood vessel remodeling; camera-type eye development; cell morphogenesis; circadian regulation of gene expression; embryonic hemopoiesis; gland development; immune system process; intracellular receptor-mediated signaling pathway; liver development; lymphocyte homeostasis; negative regulation of systemic arterial blood pressure; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; negative regulation of vasoconstriction; ovarian follicle development; patterning of blood vessels; positive regulation of cell size; positive regulation of growth rate; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; positive regulation of transcriptional preinitiation complex assembly; post-embryonic hemopoiesis; prostate gland development; regulation of B cell proliferation; regulation of blood vessel size; regulation of gene expression; regulation of transcription from RNA polymerase II promoter; regulation of transcription, DNA-dependent; reproductive structure development; response to estradiol stimulus; response to organic cyclic substance; response to toxin; response to xenobiotic stimulus; smooth muscle development; spleen development; T cell homeostasis; transcription from RNA polymerase II promoter
NCBI Summary:	binds 3-methylcholanthrene and other aromatic hydrocarbons; may play a role in bone formation [RGD, Feb 2006]
UniProt Code:	P41738
NCBI GenInfo Identifier:	29337195
NCBI Gene ID:	25690
NCBI Accession:	P41738.2
UniProt Secondary Accession:	P41738,O88930, O89105,
UniProt Related Accession:	P41738
Molecular Weight:	69,332 Da
NCBI Full Name:	Aryl hydrocarbon receptor
NCBI Synonym Full Names:	aryl hydrocarbon receptor
NCBI Official Symbol:	Ahr
NCBI Protein Information:	aryl hydrocarbon receptor
UniProt Protein Name:	Aryl hydrocarbon receptor
Protein Family:	Aldehyde reductase
UniProt Gene Name:	Ahr
UniProt Entry Name:	AHR_RAT

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!
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 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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.