LXR α, RCAS Kit, EnBio [EnBio RCAS for LXR α]

Product#: LXRA-SRC
$961.00
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LXR α, RCAS Kit, EnBio [EnBio RCAS for LXR α]

Cat. No. LXRA-SRC

Description

Liver X receptor (LXR) is a member of the nuclear receptor (NR) family. NR is one of the transcription factors that regulate target gene expression. LXR plays an important role in regulation of cholesterol and fatty acid homeostasis, therefore, the study of chemicals bind to LXR is very useful for research of drug screening.
LXR has two subtypes, LXRα and LXRβ. LXRα is expressed in restricted tissues such as liver, kidney, intestine, fat tissue and macrophages, whereas LXRβ is ubiquitously expressed. 

NR activation before gene expression consists of 3 steps. 
Step1: Ligand binding to NR induces conformational change of NR
Step2: Coactivator is recruited to NR-ligand complex
Step3: Other transcriptional factors are recruited to NR-ligand-coactivator complex

Competitive binding assay detects only the ligand-binding to NR, whereas RCAS detects NR-ligand-coactivator complex. Therefore you can predict the characteristic of your test samples more accurately using this RCAS kit. 

Principle
A peptide containing LXXLL motif of coactivator (SRC1) is immobilized on the microwell plate. The mixture of recombinant human LXRα with potentially agonistic compounds is incubated into the plate. The binding of LXRα-ligand complex to the coactivator peptide on the plate is detected by using HRP conjugated detection antibody.

The HRP activity is determined by the addition of TMB substrate solution. The reaction is stopped by addition of an acid solution and the resultant color read at 450 nm using a microwell plate spectrophotometer. The reactivity of the sample to the receptor can be determined by calculation of EC50 using the absorbance data. 
 
Assay protocol
STEP 1: Sample-LXR Reaction
1. Apply the reaction mixtures to the wells of the Coactivator binding plate (see section 7 (2)) as follows:
Add 95 μL of LXRα solution to each well followed by 5μL of agonist sample. Both positive and negative control should be measured in each assay. As a positive control, add 5 μL of T090 standard ( 0.5 mM) into the appropriate well after addition of 95 μL of LXRα solution. As a negative control, add 5 μL of DMSO into the appropriate well after addition of 95 μL of LXRα solution.
2. Cover the plate with the plate seal and incubate at room temperature (20- 28℃) for 1 hour with shaking (about 800 rpm) on a plate shaker.

STEP 2: Antibody Reaction
1. Aspirate and wash all wells as described in section 7 (2) step 2-3.
2. Pippete 100 μL of Detection antibody solution (see section 7 (1)) to each well.
3. Cover the plate with the plate seal and incubate at room temperature (20- 28℃) for 30 min with shaking (about 800 rpm) on a plate shaker.

STEP 3: Detection
1. Dispense the TMB substrate from the bottle only the amount required for the number of strips you are running, 100 μl per well. Do not use a glass pipette to measure the TMB substrate. Do not combine leftover substrate with that reserved for the second partial plate. Care must be taken to ensure that the remaining TMB substrate is not contaminated. If the substrate reagent is bright blue prior to use, it has been contaminated. DO NOT USE.
2. Aspirate and wash all wells as described in section7 (2) step 2-3.
3. Add 100 μL of TMB substrate to all wells.
4. Incubate at room temperature for 20 min (no shaking!).
5. Add 100 μL of Stop solution to all wells.
6. Read the absorbance at 450 nm. 
Background
Estrogen receptor alpha (ERa) is a member of the nuclear receptor (NR) family. NR is one of the transcription factors that regulate target gene expression. ERa plays an important role of reproduction in physiological functions, therefore, the study of chemical binding to ERa is very useful for research of endocrine disrupting chemicals (EDCs) and drug screening.

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A typical result 

  1. Dose-response curve of 17β-estradiol (E2) based on raw optical density data obtained from a 5-fold dilution series of E2 from 40 nM. 
  2. Actual Photograph of TMB color development in the wells just before adding the stop solution (the photograph was taken when the data described in panel A was measured.) 
  3. Example of data analysis using SRC1 (+) and SRC1 (-) Panel A shows OD values of the samples observed in SRC1 (+) and SRC1 (-).  Panel B indicates the difference in the observed OD values of each sample [SRC1 (+) SRC1 (-)]. The observed OD values of Sample A and Sample B are almost the same in SRC (+), but because of higher OD value of Sample B in SRC1 (-), Sample A turns out to be more active. 
  4. Typical assay data for agonist (E2, DES, Genistein, NP, BPA and Resveratrol) and antagonist (4-Hydroxytamoxifen and ICI182,780) The EC50 or IC50 value of test chemicals will be calculated by logistic 4-parameter curve fit using computer software such as Prism 4. Dose response curve of 17beta-estradiol (E2), Diethylstilbestrol (DES), Genistein, 4-Nonylphenol (NP), Bisphenol A (BPA) and Resveratrol. Dose response curve of 17beta-estradiol (E2), 4-Hydroxytamoxifen (antagonist, in combination with 2 nM E2) and ICI182,780 (antagonist, in combination with 2 nM E2)



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