Description
Human Gremlin-1 ELISA Kit
The Human Gremlin-1 ELISA Kit is specifically designed for the precise measurement of Gremlin-1 levels in human samples such as serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit offers accurate and consistent results, making it a valuable tool for various research studies.Gremlin-1 is a protein known for its role in regulating cell growth, differentiation, and development. It has been implicated in numerous disease processes, including cancer, fibrosis, and developmental disorders.
As a key biomarker, measuring Gremlin-1 levels can provide valuable insights into these conditions and aid in the development of potential therapeutic interventions.Overall, the Human Gremlin-1 ELISA Kit is essential for researchers studying the role of Gremlin-1 in disease pathogenesis and therapeutic development. Its reliability and accuracy make it a reliable choice for laboratories conducting cutting-edge research in the field of molecular biology and biomedicine.
| Product Name: | Human Gremlin-1 ELISA Kit |
| Product Code: | HUFI01783 |
| Size: | 96 Assays |
| Alias: | GREM1, Gremlin 1, DAND2, DRM, CKTSF1B1 |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human GREM1 concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 0.094ng/ml |
| Range: | 0.156-10ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human GREM1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human GREM1 in samples. | ||||||||||||||||
| |||||||||||||||||
| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human GREM1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
| |||||||||||||||||
| CV(%): | Intra-Assay: CV<8% Inter-Assay: CV<10% |
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| Sample/Standard Dilution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody(Concentrated) | 120ul | 4°C (Protect from light) |
| Antibody Dilution Buffer | 10ml | 4°C |
| HRP-Streptavidin Conjugate(SABC) | 120ul | 4°C (Protect from light) |
| SABC Dilution Buffer | 10ml | 4°C |
| TMB Substrate | 10ml | 4°C (Protect from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 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
| Uniprot | O60565 |
| UniProt Protein Function: | GREM1: Cytokine that may play an important role during carcinogenesis and metanephric kidney organogenesis, as a BMP antagonist required for early limb outgrowth and patterning in maintaining the FGF4-SHH feedback loop. Down-regulates the BMP4 signaling in a dose-dependent manner. Acts as inhibitor of monocyte chemotaxis. Interacts with SLIT1 and SLIT2 in a glycosylation- dependent manner. By high glucose through TGFB1-mediated pathways in mesangial cell. Down-regulated in tumor cell lines. Highly expressed in small intestine, fetal brain and colon. Weakly expressed in brain, ovary, prostate, pancreas and skeletal muscle. In brain found in the region localized around the internal capsule in the large subcortical nuclei, including caudate, putamen, substantia nigra, thalamus and subthalamus. Predominantly expressed in normal cells including neurons, astrocytes and fibroblasts. Belongs to the DAN family. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 15q13.3 Molecular Function:morphogen activity; protein binding; receptor agonist activity; transmembrane receptor protein tyrosine kinase activator activity; vascular endothelial growth factor receptor 2 binding Biological Process: cell migration during sprouting angiogenesis; cell morphogenesis; collagen fibril organization; determination of dorsal identity; limb development; negative regulation of apoptosis; negative regulation of BMP signaling pathway; negative regulation of bone mineralization; negative regulation of bone remodeling; negative regulation of chondrocyte differentiation; negative regulation of osteoblast proliferation; positive regulation of cell proliferation; positive regulation of receptor internalization; positive regulation of telomerase activity; positive regulation of transcription from RNA polymerase II promoter; regulation of stress-activated MAPK cascade; signal transduction |
| NCBI Summary: | This gene encodes a member of the BMP (bone morphogenic protein) antagonist family. Like BMPs, BMP antagonists contain cystine knots and typically form homo- and heterodimers. The CAN (cerberus and dan) subfamily of BMP antagonists, to which this gene belongs, is characterized by a C-terminal cystine knot with an eight-membered ring. The antagonistic effect of the secreted glycosylated protein encoded by this gene is likely due to its direct binding to BMP proteins. As an antagonist of BMP, this gene may play a role in regulating organogenesis, body patterning, and tissue differentiation. In mouse, this protein has been shown to relay the sonic hedgehog (SHH) signal from the polarizing region to the apical ectodermal ridge during limb bud outgrowth. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2010] |
| UniProt Code: | O60565 |
| NCBI GenInfo Identifier: | 62510668 |
| NCBI Gene ID: | 26585 |
| NCBI Accession: | O60565.1 |
| UniProt Secondary Accession: | O60565,Q52LV3, Q8N914, Q8N936, |
| UniProt Related Accession: | O60565 |
| Molecular Weight: | |
| NCBI Full Name: | Gremlin-1 |
| NCBI Synonym Full Names: | gremlin 1, DAN family BMP antagonist |
| NCBI Official Symbol: | GREM1 |
| NCBI Official Synonym Symbols: | DRM; HMPS; MPSH; PIG2; CRAC1; CRCS4; DAND2; HMPS1; IHG-2; DUP15q; C15DUPq; GREMLIN; CKTSF1B1 |
| NCBI Protein Information: | gremlin-1 |
| UniProt Protein Name: | Gremlin-1 |
| UniProt Synonym Protein Names: | Cell proliferation-inducing gene 2 protein; Cysteine knot superfamily 1, BMP antagonist 1; DAN domain family member 2; Down-regulated in Mos-transformed cells protein; Increased in high glucose protein 2; IHG-2 |
| Protein Family: | Gremlin |
| UniProt Gene Name: | GREM1 |
| UniProt Entry Name: | GREM1_HUMAN |
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
| Step | Protocol |
| 1. | Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells! |
| 2. | Aliquot 0.1ml standard solutions into the standard wells. |
| 3. | Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well. |
| 4. | Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells. |
| 5. | Seal the plate with a cover and incubate at 37 °C for 90 min. |
| 6. | Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2. |
| 7. | Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall. |
| 8. | Seal the plate with a cover and incubate at 37°C for 60 min. |
| 9. | Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash. |
| 10. | Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min. |
| 11. | Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min. |
| 12. | Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color. |
| 13. | Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. |
| 14. | Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. |
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. |
| McNamee et al. | A method of separating extracellular vesicles from blood shows potential clinical translation, and reveals extracellular vesicle cargo gremlin-1 as a diagnostic biomarker | Transl Oncol. 2021 | PMID: 34800917 |
