Rat GATA4 / GATA Binding Protein 4 ELISA Kit

SKU:
RTFI00802
€599

Description

ELISA Kit Technical ManualMSDS

Rat GATA4 (GATA Binding Protein 4) ELISA Kit - Information

The Assay Genie Rat GATA4 (GATA Binding Protein 4) ELISA Kit can assay for Rat GATA4 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How do our Rat GATA4 (GATA Binding Protein 4) ELISA Kits Work?

The Assay Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, Assay Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At Assay Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound Rat GATA4 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Rat GATA4 (GATA Binding Protein 4) ELISA Kit Data

Product Code

RTFI00802

UniprotP46152
Alias

GATA4, GATA binding protein 4, GATA-binding factor 4, MGC126629, transcription factor GATA-4

Detection method

Sandwich ELISA, Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of Rat GATA4 concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.313-20ng/ml

Sensitivity

0.188ng/ml

Storage

4'C for 6 months

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.

MatrixRecovery range(%)Average(%)
serum(n=5)92-10298
EDTA plasma(n=5)85-10294
UFH plasma(n=5)87-10194
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.

Sample1:21:41:8
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%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Rat GATA4 (GATA Binding Protein 4) ELISA Kit Protocol

The below protocol is a sample protocol for Rat GATA4 (GATA Binding Protein 4) ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISA Kits allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Rat GATA4 Antibody present in their sample.

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.

Assay 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 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.

Rat GATA4 (GATA Binding Protein 4) ELISA Kit components

96 Assays

Storage

ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The Assay Genie Rat GATA4 (GATA Binding Protein 4) ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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 TypeProtocol

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.

Rat GATA4 Protein Information

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  
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Additional Information

Product type:
ELISA
Reactivity:
Rat
ELISA Type:
Sandwich
Research Area:
Epigenetics and Nuclear Signaling
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