Human EZH2 ELISA Kit (HUFI03064)- High Sensitivity

Product Name:	Human EZH2 (Enhancer of zeste homolog 2) ELISA Kit
Product Code:	HUFI03064
Size:	96 Assays
Alias:	Histone-lysine N-methyltransferase EZH2, ENX-1, Enhancer of zeste homolog 2, Lysine N-methyltransferase 6, KMT6
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human EZH2 concentrations in serum plasma and other biological fluids.
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:	Matrices listed below were spiked with certain level of Human EZH2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human EZH2 in samples. Enquire for more information.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human EZH2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
CV(%):	Intra-Assay: CV<8% Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

Q15910

UniProt Protein Function:	EZH2: a histone H3 Lys 27 (H3K27) methyltransferase and Polycomb group protein with oncogenic activity. A master regulatory protein that plays a critical role in development. Catalytic subunit of the PRC2/EED-EZH2 complex, which methylates K9 and K27 of histone H3, leading to transcriptional repression of the affected target genes. PRC2 includes the Ezh2, EED, SUZ12, RBBP4 and RBBP7 and possibly AEBP2. The recruitment of PRC2 to a promoter leads to increased levels of di- and trimethylation of H3K27 (H3K27me2/3), regulating the balance between self-renewal and differentiation of embryonic stem cells (ESCs). The recruitment of PRC2 to promoters in ES cells is interdependent on Jarid 2. ESCs lacking the PRC2 component EED are deficient in Jarid2 promoter activity, while the knockdown of Jarid 2 reduces PRC2 at its target promoters. Able to mono-, di- and trimethylate K27 of histone H3 to form H3K27me1, H3K27me2 and H3K27me3, respectively. Genes repressed by the PRC2/EED-EZH2 complex include HOXC8, HOXA9, MYT1, p14ARF and retinoic acid target genes. Binds ATRX via the SET domain. The PRC2 complex may also interact with DNMT1, DNMT3A, DNMT3B and PHF1 via the EZH2 subunit and with SIRT1 via the SUZ12 subunit. Interacts with HDAC1 and HDAC2. Interacts with PRAME. Overexpressed in numerous tumor types including carcinomas of the breast, colon, larynx, lymphoma and testis. Expression decreases during senescence of embryonic fibroblasts. Expression peaks at the G1/S phase boundary. Expression is induced by E2F1, E2F2 and E2F3. Expression is reduced in cells subject to numerous types of stress including UV-, IR- and bleomycin-induced DNA damage and by activation of p53. It has been reported that phosphorylation by AKT1 on S21 reduces methyltransferase activity.
UniProt Protein Details:	Protein type:EC 2.1.1.43; Methyltransferase; Methyltransferase, protein lysine Chromosomal Location of Human Ortholog: 7q36.1 Cellular Component: ESC/E(Z) complex; nuclear chromatin; nucleoplasm; nucleus Molecular Function:chromatin binding; chromatin DNA binding; DNA binding; histone lysine N-methyltransferase activity (H3-K27 specific); histone methyltransferase activity; histone-lysine N-methyltransferase activity; protein binding; protein-lysine N-methyltransferase activity Biological Process: establishment and/or maintenance of chromatin architecture; histone methylation; negative regulation of gene expression, epigenetic; negative regulation of retinoic acid receptor signaling pathway; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; positive regulation of GTPase activity; positive regulation of MAP kinase activity; regulation of circadian rhythm; regulation of transcription, DNA-dependent Disease: Weaver Syndrome
NCBI Summary:	This gene encodes a member of the Polycomb-group (PcG) family. PcG family members form multimeric protein complexes, which are involved in maintaining the transcriptional repressive state of genes over successive cell generations. This protein associates with the embryonic ectoderm development protein, the VAV1 oncoprotein, and the X-linked nuclear protein. This protein may play a role in the hematopoietic and central nervous systems. Multiple alternatively splcied transcript variants encoding distinct isoforms have been identified for this gene. [provided by RefSeq, Feb 2011]
UniProt Code:	Q15910
NCBI GenInfo Identifier:	3334180
NCBI Gene ID:	2146
NCBI Accession:	Q15910.2
UniProt Secondary Accession:	Q15910,Q15755, Q75MG3, Q92857, Q96FI6, B2RAQ1, B3KS30 B7Z1D6, B7Z7L6,
UniProt Related Accession:	Q15910
Molecular Weight:	79,621 Da
NCBI Full Name:	Histone-lysine N-methyltransferase EZH2
NCBI Synonym Full Names:	enhancer of zeste 2 polycomb repressive complex 2 subunit
NCBI Official Symbol:	EZH2
NCBI Official Synonym Symbols:	WVS; ENX1; KMT6; WVS2; ENX-1; EZH2b; KMT6A
NCBI Protein Information:	histone-lysine N-methyltransferase EZH2
UniProt Protein Name:	Histone-lysine N-methyltransferase EZH2
UniProt Synonym Protein Names:	ENX-1; Enhancer of zeste homolog 2
Protein Family:	Histone-lysine N-methyltransferase
UniProt Gene Name:	EZH2

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
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 Âµl 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 Âµl 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 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.