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Human Mothers against decapentaplegic homolog 5 (SMAD5) ELISA Kit (HUEB2240)

SKU:
HUEB2240
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q99717
ELISA Type:
Sandwich
Reactivity:
Human
€649
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Description

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Human Mothers against decapentaplegic homolog 5 (SMAD5) ELISA Kit

The Human Mothers Against Decapentaplegic Homolog-5 (SMAD5) ELISA Kit is specifically designed for the accurate measurement of SMAD5 levels in human serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, guaranteeing precise and consistent results, making it an invaluable tool for various research purposes.SMAD5 is a critical protein involved in the TGF-β signaling pathway, regulating cell growth, differentiation, and development. Dysregulation of SMAD5 has been linked to a variety of diseases, including cancer, fibrosis, and cardiovascular disorders, underscoring its importance as a biomarker for understanding these conditions and exploring potential therapeutic interventions.

With its cutting-edge technology and comprehensive instructions, the Human SMAD5 ELISA Kit from Assay Genie provides researchers with a reliable and efficient means of quantifying SMAD5 levels, facilitating innovative discoveries and advancements in the field of molecular biology.

Product Name:Human Mothers against decapentaplegic homolog 5 (SMAD5) ELISA Kit
SKU:HUEB2240
Size:96T
Target:Human Mothers against decapentaplegic homolog 5 (SMAD5)
Synonyms:JV5-1, SMAD family member 5, SMAD 5, MAD homolog 5, MADH5
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Human
Detection Range:0.312-20ng/mL
Sensitivity:0.097ng/mL
Intra CV:Provided with the Kit
Inter CV:Provided with the Kit
Linearity:Provided with the Kit
Recovery:Provided with the Kit
Function:Transcriptional modulator activated by BMP (bone morphogenetic proteins) type 1 receptor kinase. SMAD5 is a receptor-regulated SMAD (R-SMAD).
Uniprot:Q99717
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant human Mothers against decapentaplegic homolog 5
Sub Unit:May form trimers with the co-SMAD SMAD4. Interacts with PEBP2-alpha subunit and SMURF1. Interacts with SUV39H1 and SUV39H2. Interacts (via MH2 domain) with LEMD3. Interacts with WWP1. Interacts with TMEM119 (By similarity). Interacts with ZNF8 (PubMed:12370310).
Subcellular Location:Cytoplasm Nucleus Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with SMAD4.
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:SMAD5: transcription factor phosphorylated and activated by bone morphogenetic protein receptor kinases. Phosphorylated Smads dimerize with collaborating Smad4 and are translocated into the nucleus, where they regulate transcription of target genes. Participates in a wide range of critical processes including morphogenesis, cell-fate determination, proliferation, differentiation and apoptosis.
UniProt Protein Details:

Protein type:Transcription, coactivator/corepressor; DNA-binding

Chromosomal Location of Human Ortholog: 5q31

Cellular Component: nucleoplasm; transcription factor complex; protein complex; cytoplasm; integral to membrane; intracellular; nucleus; cytosol

Molecular Function:protein binding; ubiquitin protein ligase binding; metal ion binding; receptor signaling protein activity; transcription factor activity; transforming growth factor beta receptor, pathway-specific cytoplasmic mediator activity

Biological Process: Mullerian duct regression; transcription, DNA-dependent; positive regulation of transcription, DNA-dependent; embryonic pattern specification; signal transduction; protein amino acid phosphorylation; germ cell development; BMP signaling pathway; positive regulation of osteoblast differentiation; ureteric bud development; transforming growth factor beta receptor signaling pathway; cartilage development; erythrocyte differentiation; cardiac muscle contraction; osteoblast fate commitment

NCBI Summary:The protein encoded by this gene is involved in the transforming growth factor beta signaling pathway that results in an inhibition of the proliferation of hematopoietic progenitor cells. The encoded protein is activated by bone morphogenetic proteins type 1 receptor kinase, and may be involved in cancer. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Feb 2014]
UniProt Code:Q99717
NCBI GenInfo Identifier:13959566
NCBI Gene ID:4090
NCBI Accession:Q99717.1
UniProt Secondary Accession:Q99717,O14688, Q15798, Q9UQA1,
UniProt Related Accession:Q99717
Molecular Weight:52,258 Da
NCBI Full Name:Mothers against decapentaplegic homolog 5
NCBI Synonym Full Names:SMAD family member 5
NCBI Official Symbol:SMAD5
NCBI Official Synonym Symbols:DWFC; JV5-1; MADH5
NCBI Protein Information:mothers against decapentaplegic homolog 5; SMA- and MAD-related protein 5; SMAD, mothers against DPP homolog 5; MAD, mothers against decapentaplegic homolog 5; mothers against decapentaplegic, drosophila, homolog of, 5
UniProt Protein Name:Mothers against decapentaplegic homolog 5
UniProt Synonym Protein Names:JV5-1; SMAD family member 5; SMAD 5; Smad5; hSmad5
Protein Family:SMAD5 antisense gene protein
UniProt Gene Name:SMAD5
UniProt Entry Name:SMAD5_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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