Rat Estrogen receptor beta (Esr2) ELISA Kit (RTEB1480)

Product Name:	Rat Estrogen receptor beta (Esr2) ELISA Kit
SKU:	RTEB1480
Size:	96T
Target:	Rat Estrogen receptor beta (Esr2)
Synonyms:	Nuclear receptor subfamily 3 group A member 2, ER-beta, Erbeta, Nr3a2
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.156-10ng/mL
Sensitivity:	0.078ng/mL

Intra CV:

3.9%

Inter CV:

6.6%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	88-97%	102-112%	105-113%	109-119%
EDTA Plasma(N=5)	100-110%	107-117%	91-104%	94-104%
Heparin Plasma(N=5)	104-116%	99-109%	95-107%	93-103%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	85	80-91
Plasma	87	81-93

Function:

Binds estrogens with an affinity similar to that of ER-alpha, and activates expression of reporter genes containing estrogen response elements (ERE) in an estrogen-dependent manner. Isoform 3 and isoform 4 are unable to bind DNA and activate transcription due to the truncation of the DNA binding domain. Isoform 2 shows loss of ligand binding affinity and suppresses ER-alpha and ER-beta1 mediated transcriptional activation and may act as a dominant negative regulator of estrogen action.

Uniprot:	Q62986
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Estrogen receptor beta
Sub Unit:	Binds DNA as a homodimer. Can form a heterodimer with ESR1. Interacts with NCOA1, NCOA3, NCOA5 and NCOA6 coactivators, leading to a strong increase of transcription of target genes. Interacts with PELP1, UBE1C and AKAP13. Interacts with DNTTIP2 (By similarity). Interacts with CCDC62 in the presence of estradiol/E2; this interaction seems to enhance the transcription of target genes. Interacts with PRMT2. Interacts with CCAR2 (via N-terminus) in a ligand-independent manner (By similarity). Interacts with DYX1C1.
Research Area:	Cancer
Subcellular Location:	Nucleus
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	ER-beta: a nuclear hormone receptor and transcription factor. Binds and activated by estrogen. Regulates gene expression and affects cellular proliferation and differentiation in target tissues. Binds estrogens with an affinity similar to that of ER-alpha, and activates expression of reporter genes containing estrogen response elements (ERE) in an estrogen-dependent manner. Eight alternatively-spliced isoforms have been described. Isoform beta-cx lacks ligand binding ability and has no or only very low ERE binding activity resulting in the loss of ligand-dependent transactivation ability.
UniProt Protein Details:	Protein type:DNA-binding; Nuclear receptor Cellular Component: neuron projection; cell soma; mitochondrion; perinuclear region of cytoplasm; cytoplasm; plasma membrane; perikaryon; nucleus; cilium Molecular Function:peroxisome proliferator activated receptor binding; protein binding; ligand-dependent nuclear receptor activity; estrogen receptor activity; enzyme binding; hormone binding; DNA binding; zinc ion binding; sequence-specific DNA binding; steroid hormone receptor activity; estrogen response element binding; drug binding; transcription factor activity; steroid binding Biological Process: prostate gland development; hypothalamus development; behavioral fear response; estrogen receptor signaling pathway; positive regulation of apoptosis; induction of apoptosis by hormones; negative regulation of smooth muscle cell proliferation; positive regulation of transcription, DNA-dependent; neuron migration; response to hormone stimulus; amygdala development; negative regulation of transcription from RNA polymerase II promoter; response to insecticide; uterus development; response to organic cyclic substance; response to estradiol stimulus; negative regulation of cell proliferation; learning and/or memory; ovarian follicle development; regulation of transcription, DNA-dependent; positive regulation of epidermal growth factor receptor signaling pathway; regulation of neuron apoptosis; cerebellum development; female gonad development; negative regulation of epithelial cell proliferation; steroid hormone receptor signaling pathway; response to nutrient levels; response to drug; transcription, DNA-dependent; male gonad development; vagina development; Sertoli cell proliferation; response to testosterone stimulus; Sertoli cell development; regulation of cell proliferation; response to genistein; response to ethanol; response to estrogen stimulus; positive regulation of transcription from RNA polymerase II promoter; steroid hormone mediated signaling; positive regulation of transcription factor activity; brain development; response to activity; negative regulation of behavior; response to water deprivation
NCBI Summary:	binds estrogen and mediates transcriptional activation [RGD, Feb 2006]
UniProt Code:	Q62986
NCBI GenInfo Identifier:	6978817
NCBI Gene ID:	25149
NCBI Accession:	NP_036886.1
UniProt Secondary Accession:	Q62986,O35784, O35785, O55015, O55016, O70195, Q9R185
UniProt Related Accession:	Q62986
Molecular Weight:	47,904 Da
NCBI Full Name:	estrogen receptor beta
NCBI Synonym Full Names:	estrogen receptor 2 (ER beta)
NCBI Official Symbol:	Esr2
NCBI Official Synonym Symbols:	Erb2; ERbeta; ER-beta
NCBI Protein Information:	estrogen receptor beta
UniProt Protein Name:	Estrogen receptor beta
UniProt Synonym Protein Names:	Nuclear receptor subfamily 3 group A member 2
UniProt Gene Name:	Esr2
UniProt Entry Name:	ESR2_RAT

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.