Estrogen Receptor alpha ELISA Kit

Catalog No. ABIN455080

Product Details Estrogen Receptor alpha ELISA Kit

Target: Estrogen Receptor alpha (ESR1) (Estrogen Receptor 1 (ESR1))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 78-5000 pg/mL

Minimum Detection Limit: 78 pg/mL

Application: ELISA

Purpose: This immunoassay kit allows for the specific measurement of human Estrogen Receptor,ER concentrations in cell culture supernates, serum, and plasma.

Sample Type: Cell Culture Supernatant, Serum, Plasma

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural human Estrogen Receptor.

Cross-Reactivity (Details): No significant cross-reactivity or interference was observed.

Characteristics: Homo sapiens,Human,Estrogen receptor,ER,ER-alpha,Estradiol receptor,Nuclear receptor subfamily 3 group A member 1,ESR1,ESR,NR3A1

Components: Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1x120µl), Detection Reagent B (1x120µl), Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal 2 antibody specific for Estrogen Receptor has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Estrogen Receptor present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for Estrogen Receptor is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Estrogen Receptor bound in the initial step. The color development is stopped and the intensity of the color is measured.

Reagent Preparation:

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 10 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (5 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.

Sample Collection: Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.

Assay Procedure:

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:
1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. 4
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

Calculation of Results:

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the Estrogen Receptor concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Restrictions: For Research Use only

Handling

Handling Advice: 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded. 3

Storage: 4 °C/-20 °C

Storage Comment: The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

Target Details for Estrogen Receptor alpha

Target: Estrogen Receptor alpha (ESR1) (Estrogen Receptor 1 (ESR1))

Alternative Name: ESR1 (ESR1 Products)

Synonyms: LOC398734 ELISA Kit, AA420328 ELISA Kit, AU041214 ELISA Kit, ER-alpha ELISA Kit, ER[a] ELISA Kit, ERa ELISA Kit, ERalpha ELISA Kit, ESR ELISA Kit, Estr ELISA Kit, Estra ELISA Kit, Nr3a1 ELISA Kit, NR3A1 ELISA Kit, eralpha ELISA Kit, zfER[a] ELISA Kit, ER ELISA Kit, ESRA ELISA Kit, ESTRR ELISA Kit, Era ELISA Kit, akap12 ELISA Kit, cb27 ELISA Kit, esr1 ELISA Kit, fb72g12 ELISA Kit, id:ibd1202 ELISA Kit, sb:cb27 ELISA Kit, si:ch73-192g21.1 ELISA Kit, wu:fb72g12 ELISA Kit, wu:fc21c03 ELISA Kit, Esr ELISA Kit, RNESTROR ELISA Kit, ERALPHA ELISA Kit, er ELISA Kit, esr ELISA Kit, nr3a1 ELISA Kit, XER ELISA Kit, era ELISA Kit, esra ELISA Kit, ERbeta ELISA Kit, xesr-1 ELISA Kit, xlERalpha1 ELISA Kit, xlERalpha2 ELISA Kit, ESR1 ELISA Kit, estrogen receptor 1 L homeolog ELISA Kit, estrogen receptor 1 (alpha) ELISA Kit, estrogen receptor 1 ELISA Kit, A kinase (PRKA) anchor protein 12b ELISA Kit, esr1.L ELISA Kit, Esr1 ELISA Kit, ESR1 ELISA Kit, esr1 ELISA Kit, akap12b ELISA Kit

Background: The estrogen receptor (ER) is a member of the nuclear hormone family of intracellular receptors which is activated by the hormone 17 beta-estradiol.The main function of the estrogen receptor is as a DNA binding transcription factor which regulates gene expression. However the estrogen receptor also has additional functions independent of DNA binding. There are two different forms of the estrogen receptor usually referred to as alpha and beta each encoded by a separate gene (ESR1 and ESR2 respectively). Hormone activated estrogen receptors form dimers, and since the two forms are coexpressed in many cell types, the receptors may form ERalpha (alphaalpha) or ERbeta (betabeta) homodimers or ERalphabeta (alphabeta) heterodimers.Estrogen receptor alpha and beta show significant overall sequence homology, and both are composed of seven domains.The ERalpha is found in endometrium, breast cancer cells, ovarian stroma cells and in the hypothalamus.The expression of the ERbeta protein has been documented in kidney, brain, bone, heart,lungs, intestinal mucosa, prostate, and endothelial cells. While they both bind estrogen as well as other agonists and antagonists, the two receptors have distinctly different localizations and concentrations within our body. Structural differences also exist between the two. The ER ligands tamoxifen, raloxifene, and ICI-164384 were activators with Erbeta as well as ERalpha, although the degree of agonism differed between cell types. These molecules are examples of SERM’s, selective estrogen receptor modulators. Thus, the role of estrogen complexed to ERbeta appears to be to turn off transcription of these genes, whereas the SERMs may override this blockade and activate gene transcription. Estrogen receptor structure-function is a vast topic and the subject of very active current research. Nuclear receptors are a large family of structurally related ligand-inducible transcription factors, including steroid receptors (SRs), thyroid/retinoids receptors (TR, RARs and RXRs), vitamin D receptors (VDR), LXR, PPARs, estrogen receptors (ERalpha and ERbeta), and orphan receptors for which no ligand has been yet identified. While having in common a modular structure, they are activated by distinct lipophilic small molecules such as glucocorticoids, progesterone, estrogens, retinoids, and fatty acid derivatives.All nuclear receptors have a hydrophobic pocket into which its specific ligand binds, with helix 12 (H12) being the key response element of NR's. When an agonist is bound to a NR, H12 is oriented anti-parallel to H11, capping the ligand binding pocket.

Pathways: Nuclear Receptor Transcription Pathway, EGFR Signaling Pathway, Retinoic Acid Receptor Signaling Pathway, Intracellular Steroid Hormone Receptor Signaling Pathway, Steroid Hormone Mediated Signaling Pathway, Ribonucleoprotein Complex Subunit Organization, Ribosome Assembly