Rat Smad4 / MADH4 ELISA Kit

SKU:
RTFI01134
€599

Description

ELISA Kit Technical ManualMSDS

Rat Smad4 (Mothers Against Decapentaplegic Homolog 4) ELISA Kit - Information

The Assay Genie Rat Smad4 (Mothers Against Decapentaplegic Homolog 4) ELISA Kit can assay for Rat Smad4 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How do our Rat Smad4 (Mothers Against Decapentaplegic Homolog 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 Smad4 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 Smad4 (Mothers Against Decapentaplegic Homolog 4) ELISA Kit Data

Product Code

RTFI01134

UniprotO70437
Alias

Smad4, MADH4, MAD homolog 4, Mothers against DPP homolog 4, Deletion target in pancreatic carcinoma 4, SMAD family member 4, SMAD 4, Smad4, Hsmad4

Detection method

Sandwich ELISA, Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of Rat Smad4 concentrations in serum plasma and other biological fluids.

Size

96T

Range

31.25-2000pg/ml

Sensitivity

18.75pg/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of Rat Smad4 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Smad4 in samples.

MatrixRecovery range(%)Average(%)
serum(n=5)85-10291
EDTA plasma(n=5)89-9995
UFH plasma(n=5)86-9893
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Smad4 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample1:21:41:8
serum(n=5)87-97%88-103%92-104%
EDTA plasma(n=5)87-101%83-100%83-98%
UFH plasma(n=5)81-90%89-94%82-98%
CV(%)

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

Note

For Research Use Only

Rat Smad4 (Mothers Against Decapentaplegic Homolog 4) ELISA Kit Protocol

The below protocol is a sample protocol for Rat Smad4 (Mothers Against Decapentaplegic Homolog 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 Smad4 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 Smad4 (Mothers Against Decapentaplegic Homolog 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 Smad4 (Mothers Against Decapentaplegic Homolog 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 Smad4 Protein Information

UniProt Protein Function:SMAD4: transcription factor that mediates signal transduction by the transforming growth factor superfamily. The common smad (co-smad). Binds directly to consensus DNA-binding elements in the promoters of target genes. Promotes binding of the Smad2/Smad4/Fast-1 complex to DNA and provides an activation function required for Smad1 or Smad2 to stimulate transcription.
UniProt Protein Details:

Protein type:Nuclear receptor co-regulator; Transcription, coactivator/corepressor; DNA-binding

Chromosomal Location of Human Ortholog: 18q21.1

Cellular Component: nucleoplasm; centrosome; transcription factor complex; cytoplasm; nuclear chromatin; nucleus; cytosol

Molecular Function:collagen binding; identical protein binding; protein binding; protein homodimerization activity; transforming growth factor beta receptor, common-partner cytoplasmic mediator activity; DNA binding; sequence-specific DNA binding; metal ion binding; chromatin binding; transcription factor activity

Biological Process: developmental growth; axon guidance; somatic stem cell maintenance; positive regulation of transcription, DNA-dependent; sebaceous gland development; negative regulation of transcription from RNA polymerase II promoter; palate development; BMP signaling pathway; negative regulation of cell proliferation; regulation of transforming growth factor beta receptor signaling pathway; transforming growth factor beta receptor signaling pathway; mesoderm development; neural crest cell differentiation; positive regulation of BMP signaling pathway; transcription initiation from RNA polymerase II promoter; regulation of transforming growth factor-beta2 production; transcription, DNA-dependent; regulation of binding; in utero embryonic development; neuron fate commitment; positive regulation of transforming growth factor beta receptor signaling pathway; gastrulation with mouth forming second; somite rostral/caudal axis specification; formation of anatomical boundary; SMAD protein complex assembly; endothelial cell activation; cell proliferation; ureteric bud branching; response to hypoxia; positive regulation of transcription from RNA polymerase II promoter; gene expression; regulation of hair follicle development; negative regulation of cell growth; negative regulation of transcription, DNA-dependent; negative regulation of protein catabolic process; endoderm development

Disease: Pancreatic Cancer; Juvenile Polyposis Syndrome; Myhre Syndrome; Juvenile Polyposis/hereditary Hemorrhagic Telangiectasia Syndrome

NCBI Summary:This gene encodes a member of the Smad family of signal transduction proteins. Smad proteins are phosphorylated and activated by transmembrane serine-threonine receptor kinases in response to transforming growth factor (TGF)-beta signaling. The product of this gene forms homomeric complexes and heteromeric complexes with other activated Smad proteins, which then accumulate in the nucleus and regulate the transcription of target genes. This protein binds to DNA and recognizes an 8-bp palindromic sequence (GTCTAGAC) called the Smad-binding element (SBE). The protein acts as a tumor suppressor and inhibits epithelial cell proliferation. It may also have an inhibitory effect on tumors by reducing angiogenesis and increasng blood vessel hyperpermeability. The encoded protein is a crucial component of the bone morphogenetic protein signaling pathway. The Smad proteins are subject to complex regulation by post-translational modifications. Mutations or deletions in this gene have been shown to result in pancreatic cancer, juvenile polyposis syndrome, and hereditary hemorrhagic telangiectasia syndrome. [provided by RefSeq, Aug 2017]
UniProt Code:O70437
NCBI GenInfo Identifier:4885457
NCBI Gene ID:4089
NCBI Accession:NP_005350.1
UniProt Secondary Accession:O70437,P97471, O70437,
UniProt Related Accession:Q13485
Molecular Weight:
NCBI Full Name:mothers against decapentaplegic homolog 4
NCBI Synonym Full Names:SMAD family member 4
NCBI Official Symbol:SMAD4
NCBI Official Synonym Symbols:JIP; DPC4; MADH4; MYHRS
NCBI Protein Information:mothers against decapentaplegic homolog 4
UniProt Protein Name:Mothers against decapentaplegic homolog 4
UniProt Synonym Protein Names:Deletion target in pancreatic carcinoma 4; SMAD family member 4
Protein Family:Mothers against decapentaplegic
UniProt Gene Name:SMAD4
UniProt Entry Name:SMAD4_HUMAN
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Additional Information

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