Rat CRHR1/Corticotropin-releasing factor receptor 2 ELISA Kit (RTFI00318)

Product Name:	Rat CRHR1/Corticotropin-releasing factor receptor 2 ELISA Kit
Product Code:	RTFI00318
Size:	96 Assays
Target:	Rat Crhr1
Alias:	Crhr1, Corticotropin-releasing hormone receptor 1, CRF-R-1, CRF-R1, CRFR-1, CRH-R-1, CRH-R1, seven transmembrane helix receptor, CRF1, CRFR, CRHR1f, CRH-R1h, CRHR1L, CRHRCRH-R1
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 Crhr1 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Crhr1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-104	93
EDTA plasma(n=5)	85-103	90
UFH plasma(n=5)	87-105	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Crhr1 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)	92-104%	92-105%	86-98%
EDTA plasma(n=5)	89-101%	82-100%	83-101%
UFH plasma(n=5)	80-99%	86-97%	82-100%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P35353

UniProt Protein Function:	CRF-R1: a G-protein coupled receptor that specifically binds corticotropin-releasing hormone, a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. Four splice-variant isoforms have been described.
UniProt Protein Details:	Protein type:GPCR, family 2; Membrane protein, integral; Membrane protein, multi-pass; Receptor, GPCR Chromosomal Location of Human Ortholog: 10q32.1 Cellular Component: apical part of cell; cell soma; dendrite; dendritic spine; integral to membrane; intrinsic to plasma membrane; membrane; multivesicular body; plasma membrane; trans-Golgi network; vesicle Molecular Function:corticotrophin-releasing factor receptor activity; corticotropin-releasing hormone binding; corticotropin-releasing hormone receptor activity; G-protein alpha-subunit binding; G-protein coupled receptor activity; peptide binding; protein binding; protein complex binding Biological Process: adrenal gland development; adrenocorticotropin hormone secretion; behavioral response to cocaine; behavioral response to ethanol; behavioral response to pain; elevation of cytosolic calcium ion concentration; epithelial cell differentiation; fear response; feeding behavior; G-protein signaling, adenylate cyclase activating pathway; G-protein signaling, coupled to cAMP nucleotide second messenger; G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); general adaptation syndrome, behavioral process; hypothalamus development; memory; negative regulation of epinephrine secretion; neuropeptide signaling pathway; positive regulation of cAMP biosynthetic process; positive regulation of mast cell degranulation; regulation of synaptic plasticity; response to electrical stimulus; response to hypoxia; visual learning
NCBI Summary:	This gene encodes a member of the G-protein coupled receptor family. The encoded protein is a receptor for corticotropin-releasing factor (CRH) and urocortin (UCN). The interaction of this protein with CRH and UCN triggers G-protein coupled signaling. This protein plays a pivotal role in mediating the CRH-elicited effects in depression and anxiety. Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Sep 2014]
UniProt Code:	P35353
NCBI GenInfo Identifier:	13591892
NCBI Gene ID:	58959
NCBI Accession:	NP_112261.1
UniProt Related Accession:	P35353
Molecular Weight:	47,842 Da
NCBI Full Name:	corticotropin-releasing factor receptor 1 isoform 1
NCBI Synonym Full Names:	corticotropin releasing hormone receptor 1
NCBI Official Symbol:	Crhr1
NCBI Official Synonym Symbols:	CRFR1; CRH-R 1
NCBI Protein Information:	corticotropin-releasing factor receptor 1
UniProt Protein Name:	Corticotropin-releasing factor receptor 1
UniProt Synonym Protein Names:	Corticotropin-releasing hormone receptor 1; CRH-R-1; CRH-R1
Protein Family:	Corticotropin-releasing factor receptor
UniProt Gene Name:	Crhr1

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.