Human AT1R/Angiotensin II Receptor 1 ELISA Kit (HUFI02205)

Product Name:	Human AT1R/Angiotensin II Receptor 1 ELISA Kit
Product Code:	HUFI02205
Size:	96 Assays
Alias:	ANG II R1-Ab
Detection method:	Sandwich ELISA, Double Antigen
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human ANG II R1-Ab concentrations in serum plasma and other biological fluids.
Sensitivity:	1.875ng/ml
Range:	3.125-200ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human ANG II R1-Ab R1-Ab R1-Ab and the recovery rates were calculated by comparing the measured value to the expected amount of Human ANG II R1-Ab R1-Ab R1-Ab in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-102	94
EDTA plasma(n=5)	89-103	98
UFH plasma(n=5)	88-104	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ANG II R1-Ab 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)	90-105%	95-104%	90-104%
EDTA plasma(n=5)	84-101%	86-100%	82-98%
UFH plasma(n=5)	83-97%	87-100%	85-100%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antigen (Concentrated)	120ul	4°C (Protect from light)
Antigen Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

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

Uniprot

P30556

UniProt Protein Function:	AT1: receptor for angiotensin II. Angiotensin II is a potent vasopressor hormone and a primary regulator of aldosterone secretion. It is an important effector controlling blood pressure and volume in the cardiovascular system. Mediates the major cardiovascular effects of angiotensin II. May play role in the generation of reperfusion arrhythmias following restoration of blood flow to ischemic or infarcted myocardium. Mediates its action by association with G proteins that activate a phosphatidylinositol-calcium second messenger system.
UniProt Protein Details:	Protein type:Membrane protein, integral; GPCR, family 1; Membrane protein, multi-pass; Receptor, GPCR Chromosomal Location of Human Ortholog: 3q24 Cellular Component: integral to plasma membrane; integral to membrane; plasma membrane Molecular Function:protein binding; angiotensin type II receptor activity; protein heterodimerization activity; angiotensin type I receptor activity; bradykinin receptor binding Biological Process: elevation of cytosolic calcium ion concentration during G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); regulation of vasodilation; positive regulation of cellular protein metabolic process; renin-angiotensin regulation of aldosterone production; calcium-mediated signaling; regulation of systemic arterial blood pressure by renin-angiotensin; Rho protein signal transduction; regulation of cell proliferation; G-protein coupled receptor protein signaling pathway; elevation of cytosolic calcium ion concentration; renin-angiotensin regulation of blood vessel size; regulation of vasoconstriction; regulation of inflammatory response; G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); regulation of cell growth; kidney development; positive regulation of phospholipase A2 activity; positive regulation of inflammatory response; positive regulation of NAD(P)H oxidase activity Disease: Renal Tubular Dysgenesis; Hypertension, Essential
NCBI Summary:	Angiotensin II is a potent vasopressor hormone and a primary regulator of aldosterone secretion. It is an important effector controlling blood pressure and volume in the cardiovascular system. It acts through at least two types of receptors. This gene encodes the type 1 receptor which is thought to mediate the major cardiovascular effects of angiotensin II. This gene may play a role in the generation of reperfusion arrhythmias following restoration of blood flow to ischemic or infarcted myocardium. It was previously thought that a related gene, denoted as AGTR1B, existed; however, it is now believed that there is only one type 1 receptor gene in humans. Multiple alternatively spliced transcript variants have been reported for this gene. [provided by RefSeq, Jul 2012]
UniProt Code:	P30556
NCBI GenInfo Identifier:	231519
NCBI Gene ID:	185
NCBI Accession:	P30556.1
UniProt Secondary Accession:	P30556,Q13725, Q8TBK4 ,
UniProt Related Accession:	P30556
Molecular Weight:	41kDa
NCBI Full Name:	Type-1 angiotensin II receptor
NCBI Synonym Full Names:	angiotensin II receptor, type 1
NCBI Official Symbol:	AGTR1
NCBI Official Synonym Symbols:	AT1; AG2S; AT1B; AT1R; AT1AR; AT1BR; AT2R1; HAT1R; AGTR1B
NCBI Protein Information:	type-1 angiotensin II receptor; type-1B angiotensin II receptor
UniProt Protein Name:	Type-1 angiotensin II receptor
UniProt Synonym Protein Names:	AT1AR; AT1BR; Angiotensin II type-1 receptor; AT1
Protein Family:	Type-1 angiotensin II receptor
UniProt Gene Name:	AGTR1
UniProt Entry Name:	AGTR1_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Bring all reagents and samples to room temperature 30 minutes before use. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample (diluted at least ½ with Sample Dilution Buffer) and control (zero) wells on the pre-coated plate and record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (blank) wells.
2.	Aliquot 100µl of standard solutions into the standard wells
3.	Add 100µl of Sample / Standard dilution buffer into the control (blank) well.
4.	Add 100µl of properly diluted sample (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 antigen 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 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.
Plasma	Collect 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 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 & 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 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.