Rat ER beta/ESR2/Estrogen Receptor beta ELISA Kit (RTFI00501)

Product Name:	Rat ER beta/ESR2/Estrogen Receptor beta ELISA Kit
Product Code:	RTFI00501
Size:	96 Assays
Target:	Rat Esr2
Alias:	Esr2, ESR2, NR3A2, Erb, ER-beta, ER-BETA, ESR-BETA, ESTRB, estrogen receptor 2, ER beta, estrogen receptor beta, estrogen receptor beta 4, NR3A2ESRB, Nuclear receptor subfamily 3 group A member 2
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat Esr2 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Esr2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-105	95
EDTA plasma(n=5)	85-102	92
UFH plasma(n=5)	85-104	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Esr2 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)	85-93%	89-105%	91-105%
EDTA plasma(n=5)	82-101%	85-100%	83-100%
UFH plasma(n=5)	90-100%	82-96%	81-97%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

Q62986

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

Step	Procedure
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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.