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Porcine Mothers against decapentaplegic homolog 4 (SMAD4) ELISA Kit (PREB0510)

SKU:
PREB0510
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9GKQ9
ELISA Type:
Sandwich
Reactivity:
Porcine
€699
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Description

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Porcine Mothers against decapentaplegic homolog 4 (SMAD4) ELISA Kit

The Porcine Mothers Against Decapentaplegic Homolog 4 (SMAD4) ELISA Kit is a powerful tool for researchers looking to quantify SMAD4 levels in porcine samples. This kit is specifically designed for accurate detection in porcine serum, plasma, and cell culture supernatants, providing high sensitivity and specificity for reliable and reproducible results.SMAD4 is a key player in the TGF-beta signaling pathway, regulating cell growth, differentiation, and apoptosis. Dysregulation of SMAD4 has been implicated in various diseases, including cancer, gastrointestinal disorders, and developmental abnormalities.

By accurately measuring SMAD4 levels, researchers can gain valuable insights into disease mechanisms and potential therapeutic targets.Overall, the Porcine SMAD4 ELISA Kit offers researchers a reliable and efficient method for studying SMAD4 biology in porcine models, contributing to advancements in disease research and drug development.

Product Name:Porcine Mothers against decapentaplegic homolog 4 (SMAD4) ELISA Kit
SKU:PREB0510
Size:96T
Target:Porcine Mothers against decapentaplegic homolog 4 (SMAD4)
Synonyms:SMAD family member 4, SMAD 4, MAD homolog 4, MADH4
Detection Method:ELISA
Reactivity:Pig
Intra CV:Provided with the Kit
Inter CV:Provided with the Kit
Linearity:Provided with the Kit
Recovery:Provided with the Kit
Function: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. 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. 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 (By similarity). 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.
Uniprot:Q9GKQ9
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant pig Mothers against decapentaplegic homolog 4
Sub Unit:Monomer. Heterotrimer; with a C-terminally phosphorylated R-SMAD molecule and to form the transcriptionally active SMAD2/3-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. Found in a complex with SMAD1 and YY1. Associates with ZNF423 or ZNF521 in response to BMP2 leading to activate transcription of BMP target genes. Interacts with USP9X. Interacts with RBPMS. Interacts with WWTR1 (via coiled-coil domain). Interacts with CITED1 and CITED2. Interacts with PDPK1 (via PH domain). 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. Interacts (via MH2 domain) with ZNF451 (via N-terminal zinc-finger domains) (By similarity). Identified in a complex that contains at least ZNF451, SMAD2, SMAD3 and SMAD4. Interacts weakly with ZNF8. Interacts with NUP93 and IPO7; translocates SMAD4 to the nucleus through the NPC upon BMP7 stimulation resulting in activation of SMAD4 signaling (By similarity). 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 (By similarity). Interacts with DLX1.
Research Area:Cancer
Subcellular Location:Cytoplasm Nucleus In the cytoplasm in the absence of ligand. Migration to the nucleus when complexed with R-SMAD. PDPK1 prevents its nuclear translocation.
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:Function: 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 syngernistic 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 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
UniProt Protein Details:
NCBI Summary:
UniProt Code:Q9GKQ9
NCBI GenInfo Identifier:47522958
NCBI Gene ID:397142
NCBI Accession:NP_999237.1
UniProt Secondary Accession:Q9GKQ9
UniProt Related Accession:Q9GKQ9
Molecular Weight:60,415 Da
NCBI Full Name:mothers against decapentaplegic homolog 4
NCBI Synonym Full Names:
NCBI Official Symbol:SMAD4
NCBI Official Synonym Symbols:MADH4
NCBI Protein Information:mothers against decapentaplegic homolog 4; SMAD 4; MAD homolog 4; mothers against DPP homolog 4; MAD, mothers against decapentaplegic homolog 4
UniProt Protein Name:Mothers against decapentaplegic homolog 4
UniProt Synonym Protein Names:SMAD family member 4; SMAD 4; Smad4
Protein Family:Mothers against decapentaplegic
UniProt Gene Name:SMAD4
UniProt Entry Name:SMAD4_PIG
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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