Human SMAD4 ELISA Kit (HUEB2042)- High Sensitivity

Product Name:	Human Mothers against decapentaplegic homolog 4 (SMAD4) ELISA Kit
SKU:	HUEB2042
Size:	96T
Target:	Human Mothers against decapentaplegic homolog 4 (SMAD4)
Synonyms:	Deletion target in pancreatic carcinoma 4, SMAD family member 4, SMAD 4, MAD homolog 4, DPC4, MADH4
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	15.6-1000pg/mL
Sensitivity:	8.08pg/mL

Intra CV:

7.5%

Inter CV:

11.7%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	86-98%	94-102%	108-117%	86-98%
EDTA Plasma(N=5)	104-114%	105-115%	100-110%	84-96%
Heparin Plasma(N=5)	100-110%	96-106%	102-112%	108-119%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	91	85-97
Plasma	93	87-99

Function:

In muscle physiology, plays a central role in the balance between atrophy and hypertrophy. When recruited by MSTN, promotes atrophy response via phosphorylated SMAD2/4. MSTN decrease causes SMAD4 release and subsequent recruitment by the BMP pathway to promote hypertrophy via phosphorylated SMAD1/5/8. Acts synergistically with SMAD1 and YY1 in bone morphogenetic protein (BMP)-mediated cardiac-specific gene expression. Binds to SMAD binding elements (SBEs) (5'-GTCT/AGAC-3') within BMP response element (BMPRE) of cardiac activating regions (By similarity). Common SMAD (co-SMAD) is the coactivator and mediator of signal transduction by TGF-beta (transforming growth factor). Component of the heterotrimeric SMAD2/SMAD3-SMAD4 complex that forms in the nucleus and is required for the TGF-mediated signaling. Promotes binding of the SMAD2/SMAD4/FAST-1 complex to DNA and provides an activation function required for SMAD1 or SMAD2 to stimulate transcription. Component of the multimeric SMAD3/SMAD4/JUN/FOS complex which forms at the AP1 promoter site; required for synergistic transcriptional activity in response to TGF-beta. May act as a tumor suppressor. Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator.

Uniprot:	Q13485
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Mothers against decapentaplegic homolog 4
Sub Unit:	Found in a complex with SMAD1 and YY1 (By similarity). Interacts with CITED2 (By similarity). Monomer; in the absence of TGF-beta activation. Heterodimer; on TGF-beta activation. Composed of two molecules of a C-terminally phosphorylated R-SMAD molecule, SMAD2 or SMAD3, and one molecule of SMAD4 to form the transcriptional active SMAD2/SMAD3-SMAD4 complex. Found in a ternary complex composed of SMAD4, STK11/LKB1 and STK11IP. Interacts with ATF2, COPS5, DACH1, MSG1, SKI, STK11/LKB1, STK11IP and TRIM33. Interacts with ZNF423; the interaction takes place in response to BMP2 leading to activation of transcription of BMP target genes. Interacts with ZNF521; the interaction takes place in response to BMP2 leading to activation of transcription of BMP target genes. Interacts with USP9X. Interacts (via the MH1 and MH2 domains) with RBPMS. Interacts with WWTR1 (via coiled-coil domain). Component of the multimeric complex SMAD3/SMAD4/JUN/FOS which forms at the AP1 promoter site; required for synergistic transcriptional activity in response to TGF-beta. Interacts with CITED1. Interacts with PDPK1 (via PH domain) (By similarity). Interacts with VPS39; this interaction affects heterodimer formation with SMAD3, but not with SMAD2, and leads to inhibition of SMAD3-dependent transcription activation. Interactions with VPS39 and SMAD2 may be mutually exclusive. Interacts with ZC3H3 (By similarity). Interacts (via MH2 domain) with ZNF451 (via N-terminal zinc-finger domains) (PubMed:24324267). Identified in a complex that contains at least ZNF451, SMAD2, SMAD3 and SMAD4 (PubMed:24324267). Interacts weakly with ZNF8 (PubMed:12370310). Interacts with NUP93 and IPO7; translocates SMAD4 to the nucleus through the NPC upon BMP7 stimulation resulting in activation of SMAD4 signaling (PubMed:26878725). Interacts with CREB3L1, the interaction takes place upon TGFB1 induction and SMAD4 acts as CREB3L1 coactivator to induce the expression of genes involved in the assembly of collagen extracellular matrix (PubMed:25310401). Interacts with DLX1 (PubMed:14671321).
Research Area:	Cancer
Subcellular Location:	Cytoplasm Nucleus Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with R-SMAD (PubMed:15799969). PDPK1 prevents its nuclear translocation in response to TGF-beta (PubMed:17327236).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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; DNA-binding; Transcription, coactivator/corepressor Chromosomal Location of Human Ortholog: 18q21.1 Cellular Component: nucleoplasm; centrosome; transcription factor complex; nuclear chromatin; cytoplasm; cytosol; nucleus Molecular Function:collagen binding; identical protein binding; protein binding; transforming growth factor beta receptor, common-partner cytoplasmic mediator activity; protein homodimerization 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; regulation of binding; transcription, DNA-dependent; 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; SMAD protein complex assembly; formation of anatomical boundary; endothelial cell activation; cell proliferation; ureteric bud branching; response to hypoxia; gene expression; positive regulation of transcription from RNA polymerase II promoter; negative regulation of cell growth; regulation of hair follicle development; negative regulation of protein catabolic process; negative regulation of transcription, DNA-dependent; 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 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 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, Oct 2009]
UniProt Code:	Q13485
NCBI GenInfo Identifier:	13959561
NCBI Gene ID:	4089
NCBI Accession:	Q13485.1
UniProt Secondary Accession:	Q13485,A8K405,
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; MAD homolog 4; SMAD, mothers against DPP homolog 4; deleted in pancreatic carcinoma locus 4; deletion target in pancreatic carcinoma 4; mothers against decapentaplegic, Drosophila, homolog of, 4
UniProt Protein Name:	Mothers against decapentaplegic homolog 4
UniProt Synonym Protein Names:	Deletion target in pancreatic carcinoma 4; SMAD family member 4; SMAD 4; Smad4; hSMAD4
Protein Family:	Mothers against decapentaplegic
UniProt Gene Name:	SMAD4
UniProt Entry Name:	SMAD4_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.

ELISA	Antibodies
Human Smad4 / MADH4 ELISA Kit	Anti-Smad4 Antibody (CAB18078)[KO Validated]
Human Smad4/MADH4 (Mothers Against Decapentaplegic Homolog 4 )CLIA Kit (HUES00556)	Anti-Smad4 Antibody [KO Validated] (CAB19116)
Human Smad4/MADH4 (Mothers Against Decapentaplegic Homolog 4 )ELISA Kit (HUES02057)	Anti-Smad4 Antibody (CAB5657)
Smad4 Colorimetric Cell-Based ELISA Kit	SMAD4 Antibody (PACO04272)
	MACC1 Antibody (PACO07500)