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Human Mothers against decapentaplegic homolog 6 (SMAD6) ELISA Kit

SKU:
HUEB2241
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O43541
ELISA Type:
Sandwich
Reactivity:
Human
€649
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Description

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Product Name:Human Mothers against decapentaplegic homolog 6 (SMAD6) ELISA Kit
Product Code:HUEB2241
Alias:Mothers against decapentaplegic homolog 6, MAD homolog 6, Mothers against DPP homolog 6, SMAD family member 6, SMAD 6, Smad6, hSMAD6, SMAD6, MADH6
Uniprot:O43541
Reactivity:Human
Range:Please contact us for more information
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:SMAD6: Antagonist of signaling by TGF-beta (transforming growth factor) type 1 receptor superfamily members; has been shown to inhibit selectively BMP (bone morphogenetic proteins) signaling by competing with the co-SMAD SMAD4 for receptor-activated SMAD1. SMAD6 is an inhibitory SMAD (I-SMAD) or antagonistic SMAD. Binds to regulatory elements in target promoter regions. Interacts with NEDD4L. Interacts with WWP1. Interacts with RNF111 and AXIN1. Interacts with TGF-beta type I receptor superfamily members, SMAD1, HOXC8 and HOXC9. Interacts with STAMBP and PRKX. Ubiquitous in various organs, with higher levels in lung. Isoform B is up-regulated in diseased heart tissue. Belongs to the dwarfin/SMAD family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:DNA-binding; Transcription factor

Chromosomal Location of Human Ortholog: 15q22.31

Cellular Component: transcription factor complex; protein complex; cytoplasm; cytosol; nucleus

Molecular Function:identical protein binding; protein binding; ubiquitin protein ligase binding; metal ion binding; chromatin binding; transforming growth factor beta receptor, inhibitory cytoplasmic mediator activity; transcription factor activity

Biological Process: BMP signaling pathway; fat cell differentiation; negative regulation of cell proliferation; zygotic determination of dorsal/ventral axis; transcription, DNA-dependent; ureteric bud development; response to estrogen stimulus; transforming growth factor beta receptor signaling pathway; immune response; negative regulation of transforming growth factor beta receptor signaling pathway; cell-substrate adhesion; negative regulation of apoptosis; negative regulation of BMP signaling pathway

Disease: Aortic Valve Disease 2

NCBI Summary:The protein encoded by this gene belongs to the SMAD family of proteins, which are related to Drosophila 'mothers against decapentaplegic' (Mad) and C. elegans Sma. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways. This protein functions in the negative regulation of BMP and TGF-beta/activin-signalling. Multiple transcript variants have been found for this gene.[provided by RefSeq, Sep 2014]
UniProt Code:O43541
NCBI GenInfo Identifier:115502451
NCBI Gene ID:4091
NCBI Accession:O43541.2
UniProt Related Accession:O43541
Molecular Weight:
NCBI Full Name:Mothers against decapentaplegic homolog 6
NCBI Synonym Full Names:SMAD family member 6
NCBI Official Symbol:SMAD6  
NCBI Official Synonym Symbols:AOVD2; MADH6; MADH7; HsT17432  
NCBI Protein Information:mothers against decapentaplegic homolog 6
UniProt Protein Name:Mothers against decapentaplegic homolog 6
UniProt Synonym Protein Names:SMAD family member 6; SMAD 6; Smad6; hSMAD6
Protein Family:Mothers against decapentaplegic
UniProt Gene Name:SMAD6  
UniProt Entry Name:SMAD6_HUMAN
Component Quantity (96 Assays) Storage
ELISA Microplate (Dismountable) 8×12 strips -20°C
Lyophilized Standard 2 -20°C
Sample Diluent 20ml -20°C
Assay Diluent A 10mL -20°C
Assay Diluent B 10mL -20°C
Detection Reagent A 120µL -20°C
Detection Reagent B 120µL -20°C
Wash Buffer 30mL 4°C
Substrate 10mL 4°C
Stop Solution 10mL 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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1. Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2. Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3. Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4. Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5. Repeat the wash process for five times as conducted in step 3.
6. Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7. Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8. Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9. After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

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