Mouse HDAC2 ELISA Kit (MOFI00886)- High Sensitivity

Product Name:	Mouse HDAC2 ELISA Kit
Product Code:	MOFI00886
Size:	96 Assays
Alias:	HDAC2, YAF1, HD2, histone deacetylase 2, RPD3, transcriptional regulator homolog RPD3, YAF1, YY1-associated factor 1
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse HDAC2 concentrations in serum plasma and other biological fluids.
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 Mouse HDAC2 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse HDAC2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-100	92
EDTA plasma(n=5)	86-104	96
UFH plasma(n=5)	91-103	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse HDAC2 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)	88-102%	87-103%	87-101%
EDTA plasma(n=5)	85-100%	86-100%	84-98%
UFH plasma(n=5)	83-100%	84-99%	80-100%

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

P70288

UniProt Protein Function:	HDAC2: a transcriptional regulator of the histone deacetylase family, subfamily 1. Responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4). Histone deacetylation plays a role in epigenetic repression and transcriptional regulation, cell cycle progression and developmental events. Histone deacetylases act via the formation of large multiprotein complexes.
UniProt Protein Details:	Protein type:Deacetylase; Transcription, coactivator/corepressor; EC 3.5.1.98; Nuclear receptor co-regulator Chromosomal Location of Human Ortholog: 6q21 Cellular Component: nucleoplasm; heterochromatin; transcription factor complex; protein complex; Sin3 complex; ESC/E(Z) complex; nuclear chromatin; cytoplasm; replication fork; NuRD complex; nucleus Molecular Function:deacetylase activity; NAD-dependent histone deacetylase activity (H3-K9 specific); protein binding; enzyme binding; nucleosomal DNA binding; NAD-dependent histone deacetylase activity (H3-K14 specific); sequence-specific DNA binding; heat shock protein binding; protein deacetylase activity; NAD-dependent histone deacetylase activity (H4-K16 specific); chromatin binding; histone deacetylase activity; transcription factor activity; transcription factor binding Biological Process: establishment and/or maintenance of chromatin architecture; nerve growth factor receptor signaling pathway; positive regulation of proteolysis; positive regulation of transcription, DNA-dependent; positive regulation of collagen biosynthetic process; negative regulation of transcription from RNA polymerase II promoter; regulation of gene expression, epigenetic; negative regulation of cell cycle; cardiac muscle cell development; negative regulation of gene expression, epigenetic; negative regulation of cardiac muscle cell proliferation; positive regulation of cell proliferation; dendrite development; circadian regulation of gene expression; positive regulation of oligodendrocyte differentiation; response to drug; transcription, DNA-dependent; negative regulation of transcription factor activity; hippocampus development; histone deacetylation; regulation of protein kinase B signaling cascade; response to cocaine; odontogenesis of dentine-containing teeth; chromatin remodeling; ATP-dependent chromatin remodeling; negative regulation of MHC class II biosynthetic process; maintenance of chromatin silencing; epidermal cell differentiation; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; gene expression; embryonic digit morphogenesis; negative regulation of transcription, DNA-dependent; blood coagulation; negative regulation of apoptosis
NCBI Summary:	This gene product belongs to the histone deacetylase family. Histone deacetylases act via the formation of large multiprotein complexes, and are responsible for the deacetylation of lysine residues at the N-terminal regions of core histones (H2A, H2B, H3 and H4). This protein forms transcriptional repressor complexes by associating with many different proteins, including YY1, a mammalian zinc-finger transcription factor. Thus, it plays an important role in transcriptional regulation, cell cycle progression and developmental events. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Apr 2010]
UniProt Code:	P70288
NCBI GenInfo Identifier:	293336691
NCBI Gene ID:	3066
NCBI Accession:	NP_001518.3
UniProt Secondary Accession:	P70288,P70288, B1WBY8,
UniProt Related Accession:	Q92769
Molecular Weight:
NCBI Full Name:	histone deacetylase 2
NCBI Synonym Full Names:	histone deacetylase 2
NCBI Official Symbol:	HDAC2
NCBI Official Synonym Symbols:	HD2; RPD3; YAF1; KDAC2
NCBI Protein Information:	histone deacetylase 2
UniProt Protein Name:	Histone deacetylase 2
Protein Family:	Histone deacetylase
UniProt Gene Name:	HDAC2
UniProt Entry Name:	HDAC2_HUMAN

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

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 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.