Human NCOR1 ELISA Kit (HUEB0605)- High Sensitivity

Product Name:	Human Nuclear receptor corepressor 1 (NCOR1) ELISA Kit
SKU:	HUEB0605
Size:	96T
Target:	Human Nuclear receptor corepressor 1 (NCOR1)
Synonyms:	N-CoR, KIAA1047
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	15.6-1000pg/mL
Sensitivity:	10pg/mL

Intra CV:

5.3%

Inter CV:

9.5%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	97-108%	89-98%	107-118%	101-109%
EDTA Plasma(N=5)	96-106%	111-121%	80-88%	93-102%
Heparin Plasma(N=5)	98-108%	101-112%	113-123%	108-118%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	102	96-108
Plasma	104	98-110

Function:

Mediates transcriptional repression by certain nuclear receptors. Part of a complex which promotes histone deacetylation and the formation of repressive chromatin structures which may impede the access of basal transcription factors. Participates in the transcriptional repressor activity produced by BCL6.

Uniprot:	O75376
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Nuclear receptor corepressor 1
Sub Unit:	Forms a large corepressor complex that contains SIN3A/B and histone deacetylases HDAC1 and HDAC2. This complex associates with the thyroid receptor (TR) and the retinoid acid receptor (RAR) in the absence of ligand. Interacts directly with RARA; the interaction is facilitated with RARA trimethylation. Component of the N-Cor repressor complex, at least composed of CBFA2T3, HEXIM1, NCOR1, NCOR2, HDAC3, TBL1X, TBL1XR1, CORO2A and GPS2. Interacts with ZBTB33; the interaction serves to recruit the N-CoR complex to promoter regions containing methylated CpG dinucleotides. Interacts with TRIM28 and KDM3A. Interacts (via the RD1 domain) with BAZ1A (via its N-terminal); the interaction corepresses a number of NCOR1-regulated genes. Interacts with BCL6, C1D, DACH1, HEXIM1, HDAC7, RORA, RORC, SAP30, SIAH2, SIN3A and SIN3B. May interact with DEAF1. Interacts with RXRA.
Research Area:	Stem Cells
Subcellular Location:	Nucleus
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	N-CoR1: is a protein that regulates the activity of some transcription factors, including nuclear receptors, by promoting histone deacetylation and chromatin condensation, thereby altering the accessibility of various genes to the transcriptional machinery. Is known to play a role genetic programming including that for myogenesis. Is a component of a large corepressor complex that contains SIN3A/B and histone deacetylases HDAC1 and HDAC2. This complex associates with the thyroid and the retinoic acid receptors in the absence of ligand. Interacts with the catalytic domain of HDAC9. Increased expression correlates with the loss of vitamin D responsiveness in aggressive androgen-independent prostate cancer cells. Its gene tends to be overexpressed in multiple-myeloma cell lines.
UniProt Protein Details:	Protein type:Nuclear receptor co-regulator; Transcription, coactivator/corepressor Chromosomal Location of Human Ortholog: 17p11.2 Cellular Component: histone deacetylase complex; membrane; nuclear chromatin; nucleoplasm; nucleus; Sin3 complex; spindle microtubule; transcriptional repressor complex Molecular Function:histone deacetylase binding; ligand-dependent nuclear receptor binding; nuclear hormone receptor binding; protein binding; thyroid hormone receptor binding; transcription corepressor activity Biological Process: cellular lipid metabolic process; circadian rhythm; negative regulation of JNK cascade; negative regulation of transcription from RNA polymerase II promoter; spindle assembly; transcription from RNA polymerase II promoter
NCBI Summary:	This gene encodes a protein that mediates ligand-independent transcription repression of thyroid-hormone and retinoic-acid receptors by promoting chromatin condensation and preventing access of the transcription machinery. It is part of a complex which also includes histone deacetylases and transcriptional regulators similar to the yeast protein Sin3p. This gene is located between the Charcot-Marie-Tooth and Smith-Magenis syndrome critical regions on chromosome 17. Alternate splicing results in multiple transcript variants. Pseudogenes of this gene are found on chromosomes 17 and 20.[provided by RefSeq, Jun 2010]
UniProt Code:	O75376
NCBI GenInfo Identifier:	47117817
NCBI Gene ID:	9611
NCBI Accession:	O75376.2
UniProt Secondary Accession:	O75376,Q86YY0, Q9UPV5, Q9UQ18, B3DLF8, E9PGV6,
UniProt Related Accession:	O75376
Molecular Weight:
NCBI Full Name:	Nuclear receptor corepressor 1
NCBI Synonym Full Names:	nuclear receptor corepressor 1
NCBI Official Symbol:	NCOR1
NCBI Official Synonym Symbols:	N-CoR; TRAC1; N-CoR1; hN-CoR; PPP1R109
NCBI Protein Information:	nuclear receptor corepressor 1
UniProt Protein Name:	Nuclear receptor corepressor 1
Protein Family:	Nuclear receptor corepressor
UniProt Gene Name:	NCOR1
UniProt Entry Name:	NCOR1_HUMAN

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.

ELISA	Antibodies
Human NCOR2 / Nuclear Receptor Corepressor ELISA Kit	Anti-NCOR1 Antibody (CAB7046)
NCoR1 Transcription Factor Activity Assay	RIN1 Antibody (PACO05790)
NCoR1 Colorimetric Cell-Based ELISA Kit	NCOA5 Antibody (PACO10764)
	NCK2 Antibody (PACO18281)
	NCOR1 Antibody (PACO18282)