Description
Human Growth/differentiation factor 5 (GDF5) ELISA Kit
The Human Growth Differentiation Factor 5 (GDF5) ELISA Kit is specifically designed for the precise quantification of GDF5 levels in human samples including serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for various research applications.GDF5 is a key protein that plays a crucial role in regulating growth and differentiation processes, particularly in musculoskeletal development.
Dysregulation of GDF5 has been linked to conditions such as osteoarthritis and skeletal dysplasia, highlighting its importance as a biomarker in studying these disorders and potential therapeutic interventions.Overall, the Human GDF5 ELISA Kit is a valuable tool for researchers investigating the role of GDF5 in various biological processes and disease states, offering reliable and reproducible results to advance scientific discoveries in the field.
| Product Name: | Human Growth/differentiation factor 5 (GDF5) ELISA Kit |
| SKU: | HUEB0725 |
| Size: | 96T |
| Target: | Human Growth/differentiation factor 5 (GDF5) |
| Synonyms: | Bone morphogenetic protein 14, Cartilage-derived morphogenetic protein 1, Lipopolysaccharide-associated protein 4, Radotermin, BMP-14, CDMP-1, LAP-4, GDF-5, BMP14, CDMP1 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.156-10ng/mL |
| Sensitivity: | 0.078ng/mL |
| Intra CV: | 4.6% | ||||||||||||||||||||
| Inter CV: | 8.9% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Growth factor involved in bone and cartilage formation. During cartilage development regulates differentiation of chondrogenic tissue through two pathways. Firstly, positively regulates differentiation of chondrogenic tissue through its binding of high affinity with BMPR1B and of less affinity with BMPR1A, leading to induction of SMAD1-SMAD5-SMAD8 complex phosphorylation and then SMAD protein signaling transduction (PubMed:24098149, PubMed:21976273, PubMed:15530414, PubMed:25092592). Secondly, negatively regulates chondrogenic differentiation through its interaction with NOG (PubMed:21976273). Required to prevent excessive muscle loss upon denervation. This function requires SMAD4 and is mediated by phosphorylated SMAD1/5/8 (By similarity). Binds bacterial lipopolysaccharide (LPS) and mediates LPS-induced inflammatory response, including TNF secretion by monocytes (PubMed:11276205). |
| Uniprot: | P43026 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Growth/differentiation factor 5 |
| Sub Unit: | Homodimer; disulfide-linked (By similarity). Interacts with serine proteases, HTRA1 and HTRA3 (By similarity). Following LPS binding, may form a complex with CXCR4, HSP90AA1 and HSPA8. Interacts with high affinity with NOG; inhibits chondrogenesis. Interacts with high affinity with BMPR1B and lower affinity with BMPR1A; positively regulates chondrocyte differentiation and induces SMAD dependent signaling. Interacts with FBN1 (via N-terminal domain) and FBN2 (PubMed:18339631). |
| Research Area: | Cancer |
| Subcellular Location: | Secreted Cell membrane |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | GDF5: a cytokine that is a member of the bone morphogenetic protein (BMP) family and the TGF-beta superfamily. These cytokines are characterized by a polybasic proteolytic processing site which is cleaved to produce a mature protein containing seven conserved cysteine residues. The members of this family are regulators of cell growth and differentiation in both embryonic and adult tissues. Binds to bone morphogenetic protein receptors (BMPRs), a family of transmembrane serine/threonine kinases. BMPRs are involved in bone and cartilage formation. Chondrogenic signaling is mediated by the high-affinity receptor BMPR1B. Defects in GDF5 are the cause of acromesomelic chondrodysplasia Grebe type (AMDG), acromesomelic chondrodysplasia Hunter-Thompson type (AMDH), brachydactyly type C (BDC), Du Pan syndrome (DPS), symphalangism proximal syndrome (SYM1), multiple synostoses syndrome type 2 (SYNS2), and brachydactyly type A2 (BDA2). Genetic variations in GDF5 are associated with susceptibility to osteoarthritis type 5 (OS5). Belongs to the TGF-beta family. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 20q11.2 Cellular Component: extracellular space; extracellular region; plasma membrane Molecular Function:identical protein binding; protein binding; growth factor activity; cytokine activity; transforming growth factor beta receptor binding Biological Process: hindlimb morphogenesis; extracellular matrix organization and biogenesis; regulation of multicellular organism growth; regulation of apoptosis; forelimb morphogenesis; BMP signaling pathway; positive regulation of chondrocyte differentiation; cell-cell signaling; transforming growth factor beta receptor signaling pathway; regulation of MAPKKK cascade; chondrocyte differentiation; negative regulation of neuron apoptosis; positive regulation of neuron differentiation; cell development; negative regulation of epithelial cell proliferation; embryonic limb morphogenesis; growth Disease: Chondrodysplasia, Grebe Type; Acromesomelic Dysplasia, Hunter-thompson Type; Brachydactyly, Type A1, C; Brachydactyly, Type A2; Symphalangism, Proximal, 1b; Fibular Hypoplasia And Complex Brachydactyly; Brachydactyly, Type C; Osteoarthritis Susceptibility 5; Multiple Synostoses Syndrome 2 |
| NCBI Summary: | The protein encoded by this gene is a member of the bone morphogenetic protein (BMP) family and the TGF-beta superfamily. This group of proteins is characterized by a polybasic proteolytic processing site which is cleaved to produce a mature protein containing seven conserved cysteine residues. The members of this family are regulators of cell growth and differentiation in both embryonic and adult tissues. Mutations in this gene are associated with acromesomelic dysplasia, Hunter-Thompson type; brachydactyly, type C; and chondrodysplasia, Grebe type. These associations confirm that the gene product plays a role in skeletal development. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P43026 |
| NCBI GenInfo Identifier: | 20141384 |
| NCBI Gene ID: | 8200 |
| NCBI Accession: | P43026.3 |
| UniProt Secondary Accession: | P43026,Q96SB1, E1P5Q2, |
| UniProt Related Accession: | P43026 |
| Molecular Weight: | Observed: 55kDaCalculated: 55kDa |
| NCBI Full Name: | Growth/differentiation factor 5 |
| NCBI Synonym Full Names: | growth differentiation factor 5 |
| NCBI Official Symbol: | GDF5 |
| NCBI Official Synonym Symbols: | OS5; LAP4; BDA1C; BMP14; CDMP1; LAP-4; SYM1B; SYNS2; BMP-14 |
| NCBI Protein Information: | growth/differentiation factor 5; CDMP-1; radotermin; LPS-associated protein 4; bone morphogenetic protein 14; lipopolysaccharide-associated protein 4; cartilage-derived morphogenetic protein-1 |
| UniProt Protein Name: | Growth/differentiation factor 5 |
| UniProt Synonym Protein Names: | Bone morphogenetic protein 14; BMP-14; Cartilage-derived morphogenetic protein 1; CDMP-1; Lipopolysaccharide-associated protein 4; LAP-4; LPS-associated protein 4; Radotermin |
| Protein Family: | Growth/differentiation factor |
| UniProt Gene Name: | GDF5 |
| UniProt Entry Name: | GDF5_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. |
