Human ARRB1/Beta Arrestin 1 ELISA Kit (HUFI02231)

Product Name:	Human ARRB1/Beta Arrestin 1 ELISA Kit
Product Code:	HUFI02231
Size:	96 Assays
Alias:	ARRbeta1, ARR1, ARRB1, ARB1, ARR1, arrestin 2, Arrestin beta-1, arrestin, beta 1, beta-arrestin-1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human ARRbeta1 concentrations in serum plasma and other biological fluids.
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human ARRbeta1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human ARRbeta1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	93-105	97
EDTA plasma(n=5)	88-101	95
UFH plasma(n=5)	86-96	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ARRbeta1 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)	89-105%	86-105%	88-104%
EDTA plasma(n=5)	84-100%	83-97%	90-98%
UFH plasma(n=5)	81-96%	82-100%	82-94%

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 Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody 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

P49407

UniProt Protein Function:	ARRB1: regulates G-protein coupled receptors (GPCR) signaling by mediating both receptor desensitization and resensitization processes. Binds to GRK-phosphorylated receptor and sterically preclude its coupling to the cognate G- protein; the binding appears to require receptor determinants exposed only in the active receptor conformation. Targets many receptors for internalization by acting as endocytic adapters (CLASPs, clathrin-associated sorting proteins). Internalized arrestin-receptor complexes traffic to intracellular endosomes, where they remain uncoupled from G-proteins. Two different modes of arrestin-mediated internalization occur. Beta-arrestins function as multivalent adapter proteins that can switch the GPCR from a G-protein signaling mode that transmits short-lived signals from the plasma membrane via small molecule second messengers and ion channels to a beta-arrestin signaling mode that transmits a distinct set of signals that are initiated as the receptor internalizes and transits the intracellular compartment. Also involved in regulation of receptors other than GPCRs. Involved in Toll-like receptor and IL-1 receptor signaling through the interaction with TRAF6 which prevents TRAF6 autoubiquitination and oligomerization required for activation of NF-kappa-B and JUN. Binds phosphoinositides. Binds inositolhexakisphosphate (InsP6). Involved in IL8-mediated granule release in neutrophils. Interacts with phosphorylated ADRB2 and CHRM2. Interacts with SRC (via the SH3 domain and the protein kinase domain); the interaction is independent of the phosphorylation state of SRC C-terminus. Interacts with RAF1, CHUK, IKBKB and Nik. Interacts with DVL1 and DVL2; the interaction is enhanced by DVL phosphorylation. Interacts with IGF1R. Belongs to the arrestin family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Adaptor/scaffold Chromosomal Location of Human Ortholog: 11q13 Cellular Component: basolateral plasma membrane; chromatin; coated pit; cytoplasm; cytoplasmic vesicle; cytoplasmic vesicle membrane; cytosol; dendritic spine; Golgi membrane; heterotrimeric G-protein complex; lysosomal membrane; nucleoplasm; nucleus; plasma membrane; postsynaptic density; postsynaptic membrane; pseudopodium Molecular Function:alpha-1A adrenergic receptor binding; alpha-1B adrenergic receptor binding; angiotensin receptor binding; caspase inhibitor activity; enzyme inhibitor activity; estrogen receptor binding; follicle stimulating hormone receptor binding; GTPase activator activity; histone acetyltransferase activity; insulin-like growth factor receptor binding; mitogen-activated protein kinase kinase binding; protein binding; protein phosphorylated amino acid binding; transcription factor binding; ubiquitin protein ligase binding; V2 vasopressin receptor binding Biological Process: activation of MAPK activity; blood coagulation; follicle-stimulating hormone signaling pathway; G-protein coupled receptor internalization; inhibition of NF-kappaB transcription factor; negative regulation of caspase activity; negative regulation of interleukin-6 production; negative regulation of interleukin-8 production; negative regulation of protein ubiquitination; Notch signaling pathway; phototransduction; platelet activation; positive regulation of caspase activity; positive regulation of GTPase activity; positive regulation of histone acetylation; positive regulation of peptidyl-serine phosphorylation; positive regulation of protein amino acid phosphorylation; positive regulation of protein binding; positive regulation of protein ubiquitination; positive regulation of receptor internalization; positive regulation of Rho protein signal transduction; positive regulation of transcription from RNA polymerase II promoter; post-Golgi vesicle-mediated transport; proteasomal ubiquitin-dependent protein catabolic process; protein transport; protein ubiquitination; stress fiber formation; transcription from RNA polymerase II promoter
NCBI Summary:	Members of arrestin/beta-arrestin protein family are thought to participate in agonist-mediated desensitization of G-protein-coupled receptors and cause specific dampening of cellular responses to stimuli such as hormones, neurotransmitters, or sensory signals. Arrestin beta 1 is a cytosolic protein and acts as a cofactor in the beta-adrenergic receptor kinase (BARK) mediated desensitization of beta-adrenergic receptors. Besides the central nervous system, it is expressed at high levels in peripheral blood leukocytes, and thus the BARK/beta-arrestin system is believed to play a major role in regulating receptor-mediated immune functions. Alternatively spliced transcripts encoding different isoforms of arrestin beta 1 have been described. [provided by RefSeq, Jan 2011]
UniProt Code:	P49407
NCBI GenInfo Identifier:	20141238
NCBI Gene ID:	408
NCBI Accession:	P49407.2
UniProt Secondary Accession:	P49407,O75625, O75630, Q2PP20, Q9BTK8, B6V9G8,
UniProt Related Accession:	P49407
Molecular Weight:	46,309 Da
NCBI Full Name:	Beta-arrestin-1
NCBI Synonym Full Names:	arrestin beta 1
NCBI Official Symbol:	ARRB1
NCBI Official Synonym Symbols:	ARB1; ARR1
NCBI Protein Information:	beta-arrestin-1
UniProt Protein Name:	Beta-arrestin-1
UniProt Synonym Protein Names:	Arrestin beta-1
Protein Family:	Beta-arrestin
UniProt Gene Name:	ARRB1
UniProt Entry Name:	ARRB1_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.

Step	Protocol
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 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.