Rat GATA4/GATA Binding Protein 4 ELISA Kit (RTFI00802)

Product Name:	Rat GATA4/GATA Binding Protein 4 ELISA Kit
Product Code:	RTFI00802
Size:	96 Assays
Target:	Rat GATA4
Alias:	GATA4, GATA binding protein 4, GATA-binding factor 4, MGC126629, transcription factor GATA-4
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat GATA4 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat GATA4 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	92-102	98
EDTA plasma(n=5)	85-102	94
UFH plasma(n=5)	87-101	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat GATA4 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)	91-99%	86-103%	85-101%
EDTA plasma(n=5)	84-101%	89-99%	86-100%
UFH plasma(n=5)	80-97%	93-98%	92-97%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P46152

UniProt Protein Function:	GATA4: a conserved and ubiquitous member of the GATA family of zinc-finger transcription factors. Members of this family recognize the GATA motif which is present in the promoters of many genes. This protein is thought to regulate genes involved in embryogenesis and in myocardial differentiation and function. May regulate a set of cardiac-specific genes and play a crucial role in cardiogenesis. Defects are a cause of atrial septal defect 2 (ASD2). ASD2 is an autosomal dominant condition with atrial septal defect and other congenital heart disease but no conduction defects or noncardiac abnormalities.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Transcription factor Chromosomal Location of Human Ortholog: 15p12 Cellular Component: nuclear body; nuclear chromatin; nucleus; RNA polymerase II transcription factor complex; transcription factor complex Molecular Function:activating transcription factor binding; chromatin binding; co-SMAD binding; DNA binding; DNA binding transcription factor activity; NFAT protein binding; protein kinase binding; sequence-specific DNA binding; transcription coactivator activity; transcription factor activity, RNA polymerase II distal enhancer sequence-specific binding; transcription factor binding; transcription regulatory region DNA binding; zinc ion binding Biological Process: atrial septum primum morphogenesis; atrial septum secundum morphogenesis; atrioventricular valve formation; atrioventricular valve morphogenesis; BMP signaling pathway; cardiac muscle cell differentiation; cardiac muscle hypertrophy in response to stress; cardiac muscle tissue development; cardiac right ventricle morphogenesis; cardiac septum development; cell-cell signaling; cellular response to follicle-stimulating hormone stimulus; cellular response to glucose stimulus; cellular response to gonadotropin stimulus; embryonic digestive tract morphogenesis; embryonic foregut morphogenesis; embryonic heart tube anterior/posterior pattern formation; embryonic heart tube development; embryonic morphogenesis; endocardial cushion development; endoderm formation; epithelial cell fate commitment; gastrulation with mouth forming second; heart development; heart looping; heart morphogenesis; in utero embryonic development; male gonad development; negative regulation of apoptosis; negative regulation of apoptotic signaling pathway; negative regulation of autophagy; negative regulation of cardiac muscle cell apoptotic process; negative regulation of connective tissue replacement; negative regulation of gene expression; positive regulation of angiogenesis; positive regulation of BMP signaling pathway; positive regulation of cardiac muscle cell proliferation; positive regulation of cardioblast differentiation; positive regulation of cell cycle; positive regulation of ERK1 and ERK2 cascade; positive regulation of gene expression; positive regulation of protein phosphorylation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-templated; positive regulation of vascular endothelial growth factor production; regulation of cardiac muscle cell contraction; regulation of cardiac muscle cell proliferation; regulation of gene expression; regulation of protein kinase B signaling; regulation of transcription from RNA polymerase II promoter; regulation of transcription, DNA-templated; response to drug; response to estrogen; response to mechanical stimulus; response to retinoic acid; response to vitamin A; seminiferous tubule development; Sertoli cell differentiation; spermatogenesis; transcription from RNA polymerase II promoter; tube morphogenesis; ventricular cardiac muscle tissue development; ventricular septum development; Wnt receptor signaling pathway through beta-catenin
NCBI Summary:	plays a role in regulation of cardiac-myocyte specific gene expression; interacts with dHAND (HAND2) to activate transcription of several cardiac-specific genes [RGD, Feb 2006]
UniProt Code:	P46152
NCBI GenInfo Identifier:	25282465
NCBI Gene ID:	54254
NCBI Accession:	NP_653331.1
UniProt Related Accession:	P46152
Molecular Weight:	44,597 Da
NCBI Full Name:	transcription factor GATA-4
NCBI Synonym Full Names:	GATA binding protein 4
NCBI Official Symbol:	Gata4
NCBI Protein Information:	transcription factor GATA-4
UniProt Protein Name:	Transcription factor GATA-4
UniProt Synonym Protein Names:	DNA-binding protein GATA-GT2; GATA-binding factor 4
Protein Family:	GATA transcription factor
UniProt Gene Name:	Gata4

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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.