Rat Estrogen receptor (Esr1) ELISA Kit (RTEB0605)

Product Name:	Rat Estrogen receptor (Esr1) ELISA Kit
SKU:	RTEB0605
Size:	96T
Target:	Rat Estrogen receptor (Esr1)
Synonyms:	ER-alpha, Estradiol receptor, Nuclear receptor subfamily 3 group A member 1, ER, Esr, Estr, Nr3a1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.312-20ng/mL
Sensitivity:	0.158ng/mL

Intra CV:

5.8%

Inter CV:

9.3%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	109-120%	102-113%	99-111%	86-96%
EDTA Plasma(N=5)	93-102%	94-103%	108-117%	98-107%
Heparin Plasma(N=5)	108-117%	102-114%	83-93%	98-110%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	97	91-103
Plasma	99	93-105

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. Essential for MTA1-mediated transcriptional regulation of BRCA1 and BCAS3.

Uniprot:	P06211
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Estrogen receptor
Sub Unit:	Interacts with BCAS3. Binds DNA as a homodimer (By similarity). Can form a heterodimer with ESR2 (By similarity). Interacts with coactivator NCOA5. Interacts with NCOA7; the interaction is ligand-inducible. Interacts with AKAP13, CUEDC2, HEXIM1, KDM5A, MAP1S, PELP1, SMARD1, and UBE1C. Interacts with MUC1; the interaction is stimulated by 7 beta-estradiol (E2) and enhances ERS1-mediated transcription. Interacts with DNTTIP2, and UIMC1. Interacts with KMT2D/MLL2. Interacts with ATAD2; the interaction is enhanced by estradiol. Interacts with KIF18A and LDB1. Interacts with RLIM (via its C-terminus). Interacts with MACROD1. Interacts with SH2D4A and PLCG. Interacts with SH2D4A; the interaction blocks binding to PLCG and inhibits estrogen-induced cell proliferation. Interacts with DYNLL1. Interacts with CCDC62; the interaction requires estradiol and appears 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 PIK3R2, SRC and PTK2/FAK1. Interacts with RBFOX2. Interacts with EP300; the interaction is estrogen-dependent and enhanced by CITED1. Interacts with CITED1; the interaction is estrogen-dependent (By similarity). Interacts with FAM120B, FOXL2, PHB2 and SLC30A9. Interacts with coactivators NCOA3 and NCOA6. 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 (By similarity). Interacts with TRIP4 (ufmylated); estrogen dependent (By similarity). 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 (By similarity). Interacts with SFR1 in a ligand-dependent and -independent manner (By similarity). Interacts with DCAF13, LATS1 and VPRBP; regulates ESR1 ubiquitination and ubiquitin-mediated proteasomal degradation. 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 Cytoplasm 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:	ER-alpha: a nuclear hormone receptor and transcription factor. Regulates gene expression and affects cellular proliferation and differentiation in target tissues. Two splice-variant isoforms have been described.
UniProt Protein Details:	Protein type:DNA-binding; Nuclear receptor Chromosomal Location of Human Ortholog: 1q11 Cellular Component: cytoplasm; Golgi apparatus; mitochondrion; neuron projection; nuclear chromatin; nucleus; perikaryon; perinuclear region of cytoplasm; plasma membrane; protein complex; T-tubule; terminal bouton Molecular Function:ATPase binding; beta-catenin binding; chromatin binding; DNA binding; DNA binding transcription factor activity; enzyme binding; estrogen receptor activity; estrogen receptor binding; estrogen response element binding; hormone binding; identical protein binding; ligand-dependent nuclear receptor activity; protein binding; protein complex binding; protein kinase binding; sequence-specific DNA binding; steroid binding; steroid hormone receptor activity; TATA-binding protein binding; transcription factor binding; type 1 metabotropic glutamate receptor binding; zinc ion binding Biological Process: androgen metabolic process; antral ovarian follicle growth; decidualization; elevation of cytosolic calcium ion concentration; epithelial cell development; intracellular estrogen receptor signaling pathway; male gonad development; negative regulation of glucose import; negative regulation of I-kappaB kinase/NF-kappaB signaling; negative regulation of smooth muscle cell proliferation; negative regulation of transcription factor activity; negative regulation of transcription from RNA polymerase II promoter; osteoblast development; phospholipase C-activating G-protein coupled receptor signaling pathway; positive regulation of epidermal growth factor receptor signaling pathway; positive regulation of epithelial cell proliferation; positive regulation of fibroblast proliferation; positive regulation of nitric oxide biosynthetic process; positive regulation of nitric-oxide synthase activity; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-templated; positive regulation of transcriptional preinitiation complex assembly; regulation of apoptosis; regulation of inflammatory response; regulation of neuron apoptosis; regulation of toll-like receptor signaling pathway; regulation of transcription, DNA-templated; response to estradiol; response to estrogen; Sertoli cell development; Sertoli cell proliferation; stem cell differentiation; steroid hormone mediated signaling; steroid hormone receptor signaling pathway; transcription from RNA polymerase II promoter; transcription, DNA-dependent; uterus development; vagina development
NCBI Summary:	acts as a transcriptional activator when bound to estrogen; may play a role in myocardial regulation [RGD, Feb 2006]
UniProt Code:	P06211
NCBI GenInfo Identifier:	6978815
NCBI Gene ID:	24890
NCBI Accession:
UniProt Related Accession:	P06211
Molecular Weight:
NCBI Full Name:	estrogen receptor
NCBI Synonym Full Names:	estrogen receptor 1
NCBI Official Symbol:	Esr1
NCBI Official Synonym Symbols:	Esr; ER-alpha; RNESTROR
NCBI Protein Information:	estrogen receptor
UniProt Protein Name:	Estrogen receptor
UniProt Synonym Protein Names:	ER-alpha; Estradiol receptor; Nuclear receptor subfamily 3 group A member 1
Protein Family:	Regulatory protein
UniProt Gene Name:	Esr1

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.

Rat Estrogen receptor (Esr1) ELISA Kit (RTEB0605)

Description