Human Estrogen receptor (ESR1) ELISA Kit- High Sensitivity

Product Name:	Human Estrogen receptor (ESR1) ELISA Kit
SKU:	HUEB1024
Size:	96T
Target:	Human Estrogen receptor (ESR1)
Synonyms:	ER-alpha, Estradiol receptor, Nuclear receptor subfamily 3 group A member 1, ER, ESR, NR3A1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	78-5000pg/mL
Sensitivity:	32pg/mL

Intra CV:

3.2%

Inter CV:

7.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	106-116%	84-94%	99-111%	107-119%
EDTA Plasma(N=5)	94-104%	93-105%	98-110%	88-97%
Heparin Plasma(N=5)	81-93%	96-105%	96-109%	93-104%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	100	94-106
Plasma	102	96-108

Function:

Nuclear hormone receptor. The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Ligand-dependent nuclear transactivation involves either direct homodimer binding to a palindromic estrogen response element (ERE) sequence or association with other DNA-binding transcription factors, such as AP-1/c-Jun, c-Fos, ATF-2, Sp1 and Sp3, to mediate ERE-independent signaling. Ligand binding induces a conformational change allowing subsequent or combinatorial association with multiprotein coactivator complexes through LXXLL motifs of their respective components. Mutual transrepression occurs between the estrogen receptor (ER) and NF-kappa-B in a cell-type specific manner. Decreases NF-kappa-B DNA-binding activity and inhibits NF-kappa-B-mediated transcription from the IL6 promoter and displace RELA/p65 and associated coregulators from the promoter. Recruited to the NF-kappa-B response element of the CCL2 and IL8 promoters and can displace CREBBP. Present with NF-kappa-B components RELA/p65 and NFKB1/p50 on ERE sequences. Can also act synergistically with NF-kappa-B to activate transcription involving respective recruitment adjacent response elements; the function involves CREBBP. Can activate the transcriptional activity of TFF1. Also mediates membrane-initiated estrogen signaling involving various kinase cascades. Isoform 3 is involved in activation of NOS3 and endothelial nitric oxide production. Isoforms lacking one or several functional domains are thought to modulate transcriptional activity by competitive ligand or DNA binding and/or heterodimerization with the full length receptor. Essential for MTA1-mediated transcriptional regulation of BRCA1 and BCAS3. Isoform 3 can bind to ERE and inhibit isoform 1.

Uniprot:	P03372
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Estrogen receptor
Sub Unit:	Binds DNA as a homodimer. Can form a heterodimer with ESR2. Isoform 3 can probably homodimerize or heterodimerize with isoform 1 and ESR2. Interacts with FOXC2, MAP1S, SLC30A9, UBE1C and NCOA3 coactivator (By similarity). Interacts with EP300; the interaction is estrogen-dependent and enhanced by CITED1. Interacts with CITED1; the interaction is estrogen-dependent. Interacts with NCOA5 and NCOA6 coactivators. Interacts with NCOA7; the interaction is a ligand-inducible. Interacts with PHB2, PELP1 and UBE1C. Interacts with AKAP13. Interacts with CUEDC2. Interacts with KDM5A. Interacts with SMARD1. Interacts with HEXIM1. Interacts with PBXIP1. Interaction with MUC1 is stimulated by 7 beta-estradiol (E2) and enhances ERS1-mediated transcription. Interacts with DNTTIP2, FAM120B and UIMC1. Interacts with isoform 4 of TXNRD1. Interacts with KMT2D/MLL2. Interacts with ATAD2 and this interaction is enhanced by estradiol. Interacts with KIF18A and LDB1. Interacts with RLIM (via C-terminus). Interacts with MACROD1. Interacts with SH2D4A and PLCG. Interaction with SH2D4A blocks binding to PLCG and inhibits estrogen-induced cell proliferation. Interacts with DYNLL1. Interacts with CCDC62 in the presence of estradiol/E2; this interaction seems to enhance the transcription of target genes. Interacts with NR2C1; the interaction prevents homodimerization of ESR1 and suppresses its transcriptional activity and cell growth. Interacts with DYX1C1. Interacts with PRMT2. Interacts with PI3KR1 or PI3KR2, SRC and PTK2/FAK1. Interacts with RBFOX2. Interacts with STK3/MST2 only in the presence of SAV1 and vice-versa. Binds to CSNK1D. Interacts with NCOA2; NCOA2 can interact with ESR1 AF-1 and AF-2 domains simultaneously and mediate their transcriptional synergy. Interacts with DDX5. Interacts with NCOA1; the interaction seems to require a self-association of N-terminal and C-terminal regions. Interacts with ZNF366, DDX17, NFKB1, RELA, SP1 and SP3. Interacts with NRIP1 (By similarity). Interacts with GPER1; the interaction occurs in an estrogen-dependent manner. Interacts with CLOCK and the interaction is stimulated by estrogen. Interacts with BCAS3. Interacts with TRIP4 (ufmylated); estrogen dependent. Interacts with LMTK3; the interaction phosphorylates ESR1 (in vitro) and protects it against proteasomal degradation. Interacts with CCAR2 (via N-terminus) in a ligand-independent manner. Interacts with ZFHX3. Interacts with SFR1 in a ligand-dependent and -independent manner (PubMed:23874500). Interacts with DCAF13, LATS1 and VPRBP; regulates ESR1 ubiquitination and ubiquitin-mediated proteasomal degradation (PubMed:28068668). Interacts (via DNA-binding domain) with POU4F2 (C-terminus); this interaction increases the estrogen receptor ESR1 transcriptional activity in a DNA- and ligand 17-beta-estradiol-independent manner.
Research Area:	Cancer
Subcellular Location:	Nucleus Golgi apparatus Cell membrane Colocalizes with ZDHHC7 and ZDHHC21 in the Golgi apparatus where most probably palmitoylation occurs. Associated with the plasma membrane when palmitoylated.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	Function: Nuclear hormone receptor. The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Ligand-dependent nuclear transactivation involves either direct homodimer binding to a palindromic estrogen response element (ERE) sequence or association with other DNA-binding transcription factors, such as AP-1/c-Jun, c-Fos, ATF-2, Sp1 and Sp3, to mediate ERE-independent signaling. Ligand binding induces a conformational change allowing subsequent or combinatorial association with multiprotein coactivator complexes through LXXLL motifs of their respective components. Mutual transrepression occurs between the estrogen receptor (ER) and NF-kappa-B in a cell-type specific manner. Decreases NF-kappa-B DNA-binding activity and inhibits NF-kappa-B-mediated transcription from the IL6 promoter and displace RELA/p65 and associated coregulators from the promoter. Recruited to the NF-kappa-B response element of the CCL2 and IL8 promoters and can displace CREBBP. Present with NF-kappa-B components RELA/p65 and NFKB1/p50 on ERE sequences. Can also act synergistically with NF-kappa-B to activate transcription involving respective recruitment adjacent response elements; the function involves CREBBP. Can activate the transcriptional activity of TFF1. Also mediates membrane-initiated estrogen signaling involving various kinase cascades. Isoform 3 is involved in activation of NOS3 and endothelial nitric oxide production. Isoforms lacking one or several functional domains are thought to modulate transcriptional activity by competitive ligand or DNA binding and/or heterodimerization with the full length receptor. Isoform 3 can bind to ERE and inhibit isoform 1. Ref.17 Ref.19 Ref.24 Ref.30 Ref.31 Ref.33 Ref.36 Ref.44 Ref.46 Ref.47 Ref.48 Ref.52 Ref.63 Ref.65 Ref.70 Ref.75 Ref.77 Ref.78 Ref.80
UniProt Protein Details:	Subunit structure: Binds DNA as a homodimer. Can form a heterodimer with ESR2. Isoform 3 can probably homodimerize or heterodimerize with isoform 1 and ESR2. Interacts with FOXC2, MAP1S, SLC30A9, UBE1C and NCOA3 coactivator By similarity. Interacts with EP300; the interaction is estrogen-dependent and enhanced by CITED1. Interacts with CITED1; the interaction is estrogen-dependent. Interacts with NCOA5 and NCOA6 coactivators. Interacts with NCOA7; the interaction is a ligand-inducible. Interacts with PHB2, PELP1 and UBE1C. Interacts with AKAP13. Interacts with CUEDC2. Interacts with KDM5A. Interacts with SMARD1. Interacts with HEXIM1. Interacts with PBXIP1. Interaction with MUC1 is stimulated by 7 beta-estradiol (E2) and enhances ERS1-mediated transcription. Interacts with DNTTIP2, FAM120B and UIMC1. Interacts with isoform 4 of TXNRD1. Interacts with KMT2D/MLL2. Interacts with ATAD2 and this interaction is enhanced by estradiol. Interacts with KIF18A and LDB1. Interacts with RLIM (via C-terminus). Interacts with MACROD1. Interacts with SH2D4A and PLCG. Interaction with SH2D4A blocks binding to PLCG and inhibits estrogen-induced cell proliferation. Interacts with DYNLL1. Interacts with CCDC62 in the presence of estradiol/E2; this interaction seems to enhance the transcription of target genes. Interacts with NR2C1; the interaction prevents homodimerization of ESR1 and suppresses its transcriptional activity and cell growth. Interacts with DYX1C1. Interacts with PRMT2. Interacts with PI3KR1 or PI3KR2, SRC and PTK2/FAK1. Interacts with RBFOX2. Interacts with STK3/MST2 only in the presence of SAV1 and vice-versa. Binds to CSNK1D. Interacts with NCOA2; NCOA2 can interact with ESE1 AF-1 and AF-2 domains simultaneously and mediate their transcriptional synergy. Interacts with DDX5. Interacts with NCOA1; the interaction seems to require a self-association of N-terminal and C-terminal regions. Interacts with ZNF366, DDX17, NFKB1, RELA, SP1 and SP3. Interacts with NRIP1 By similarity. Interacts with GPER1; the interaction occurs in an estrogen-dependent manner. Ref.18 Ref.19 Ref.21 Ref.24 Ref.25 Ref.27 Ref.28 Ref.29 Ref.30 Ref.31 Ref.32 Ref.33 Ref.34 Ref.35 Ref.36 Ref.37 Ref.38 Ref.39 Ref.40 Ref.41 Ref.42 Ref.43 Ref.45 Ref.48 Ref.50 Ref.51 Ref.53 Ref.54 Ref.55 Ref.56 Ref.57 Ref.58 Ref.59 Ref.60 Ref.61 Ref.62 Ref.64 Ref.65 Ref.66 Ref.67 Ref.68 Ref.69 Ref.70 Ref.71 Ref.72 Ref.73 Ref.74 Ref.76 Ref.91 Subcellular location: Isoform 1: Nucleus. Cytoplasm. Cell membrane; Peripheral membrane protein; Cytoplasmic side. Note: A minor fraction is associated with the inner membrane. Ref.6 Ref.22 Ref.48 Ref.59 Ref.64 Ref.77 Ref.79Isoform 3: Nucleus. Cytoplasm. Cell membrane; Peripheral membrane protein; Cytoplasmic side. Cell membrane; Single-pass type I membrane protein. Note: Associated with the inner membrane via palmitoylation Probable. At least a subset exists as a transmembrane protein with a N-terminal extracellular domain. Ref.6 Ref.22 Ref.48 Ref.59 Ref.64 Ref.77 Ref.79Nucleus. Golgi apparatus. Cell membrane. Note: Colocalizes with ZDHHC7 and ZDHHC21 in the Golgi apparatus where most probably palmitoylation occurs. Associated with the plasma membrane when palmitoylated. Ref.6 Ref.22 Ref.48 Ref.59 Ref.64 Ref.77 Ref.79 Tissue specificity: Widely expressed. Isoform 3 is not expressed in the pituitary gland. Ref.19 Domain: Composed of three domains: a modulating N-terminal domain, a DNA-binding domain and a C-terminal ligand-binding domain. The modulating domain, also known as A/B or AF-1 domain has a ligand-independent transactivation function. The C-terminus contains a ligand-dependent transactivation domain, also known as E/F or AF-2 domain which overlaps with the ligand binding domain. AF-1 and AF-2 activate transcription independently and synergistically and act in a promoter- and cell-specific manner. AF-1 seems to provide the major transactivation function in differentiated cells. Post-translational modification: Phosphorylated by cyclin A/CDK2 and CK1. Phosphorylation probably enhances transcriptional activity. Self-association induces phosphorylation. Dephosphorylation at Ser-118 by PPP5C inhibits its transactivation activity. Ref.10 Ref.14 Ref.16 Ref.26 Ref.46 Ref.72Glycosylated; contains N-acetylglucosamine, probably O-linked. Ref.23Ubiquitinated. Deubiquitinated by OTUB1. Ref.71Dimethylated by PRMT1 at Arg-260. The methylation may favor cytoplasmic localization. Ref.64Palmitoylated (isoform 3) Not biotinylated (isoform 3) Ref.22 Ref.79Palmitoylated by ZDHHC7 and ZDHHC21. Palmitoylation is required for plasma membrane targeting and for rapid intracellular signaling via ERK and AKT kinases and cAMP generation, but not for signaling mediated by the nuclear hormone receptor. Ref.22 Ref.79 Polymorphism: Genetic variations in ESR1 are correlated with bone mineral density (BMD). Low BMD is a risk factor for osteoporotic fracture. Osteoporosis is characterized by reduced bone mineral density, disrutption of bone microarchitecture, and the alteration of the amount and variety of non-collagenous proteins in bone. Osteoporotic bones are more at risk of fracture. Involvement in Disease: Estrogen resistance (ESTRR) [MIM:615363]: A disorder characterized by partial or complete resistance to estrogens, in the presence of elevated estrogen serum levels. Clinical features include absence of the pubertal growth spurt, delayed bone maturation, unfused epiphyses, reduced bone mineral density, osteoporosis, continued growth into adulthood and very tall adult stature. Glucose intolerance, hyperinsulinemia and lipid abnormalities may also be present.Note: The disease is caused by mutations affecting the gene represented in this entry. Ref.88 Ref.93 Miscellaneous: Selective estrogen receptor modulators (SERMs), such as tamoxifen, raloxifene, toremifene, lasofoxifene, clomifene, femarelle and ormeloxifene, have tissue selective agonistic and antagonistic effects on the estrogen receptor (ER). They interfere with the ER association with coactivators or corepressors, mainly involving the AF-2 domain. Sequence similarities: Belongs to the nuclear hormone receptor family. NR3 subfamily.Contains 1 nuclear receptor DNA-binding domain.
NCBI Summary:	This gene encodes an estrogen receptor, a ligand-activated transcription factor composed of several domains important for hormone binding, DNA binding, and activation of transcription. The protein localizes to the nucleus where it may form a homodimer or a heterodimer with estrogen receptor 2. Estrogen and its receptors are essential for sexual development and reproductive function, but also play a role in other tissues such as bone. Estrogen receptors are also involved in pathological processes including breast cancer, endometrial cancer, and osteoporosis. Alternative splicing results in several transcript variants, which differ in their 5' UTRs and use different promoters. [provided by RefSeq, Jul 2008]
UniProt Code:	P03372
NCBI GenInfo Identifier:	544257
NCBI Gene ID:	2099
NCBI Accession:	P03372.2
UniProt Secondary Accession:	P03372,Q13511, Q14276, Q5T5H7, Q6MZQ9, Q9NU51, Q9UDZ7 Q9UIS7,
UniProt Related Accession:	P03372
Molecular Weight:
NCBI Full Name:	Estrogen receptor
NCBI Synonym Full Names:	estrogen receptor 1
NCBI Official Symbol:	ESR1
NCBI Official Synonym Symbols:	ER; ESR; Era; ESRA; ESTRR; NR3A1
NCBI Protein Information:	estrogen receptor; ER-alpha; estradiol receptor; estrogen nuclear receptor alpha; estrogen receptor alpha E1-E2-1-2; estrogen receptor alpha E1-N2-E2-1-2; estrogen receptor alpha delta 4 +49 isoform; nuclear receptor subfamily 3 group A member 1; estrogen receptor alpha 3,4,5,6,7/822 isoform; estrogen receptor alpha delta 4,5,6,7/654 isoform; estrogen receptor alpha delta 4,5,6,7,8/901 isoform; estrogen receptor alpha delta 3,4,5,6,7/819-2 isoform; estrogen receptor alpha delta 3,4,5,6,7,8*/941 isoform
UniProt Protein Name:	Estrogen receptor
UniProt Synonym Protein Names:	ER-alpha; Estradiol receptor; Nuclear receptor subfamily 3 group A member 1
Protein Family:	Erbin
UniProt Gene Name:	ESR1
UniProt Entry Name:	ESR1_HUMAN

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

ELISA	Antibodies
Human ER alpha (Estrogen Receptor Alpha) CLIA Kit (HUES00662)	Anti-Estrogen Receptor alpha Antibody (CAB0296)
Human ER alpha (Estrogen Receptor Alpha) ELISA Kit (HUES02220)	Anti-ESR1 Antibody (CAB12976)
Estrogen Receptor-alpha (Phospho-Ser118) Transcription Factor Activity Assay	Anti-ESR1 Antibody (CAB16668)
Estrogen Receptor-alpha Transcription Factor Activity Assay	Anti-Phospho-Estrogen receptor-S118 Antibody (CABP0146)
	Anti-Phospho-Estrogen receptor-S106 Antibody (CABP0347)