Description
Human Collagen alpha-1 (VII)chain/COL7A1 ELISA Kit
The Human Collagen Alpha 1 (VII) Chain (COL7A1) ELISA Kit is a powerful tool for the quantitative measurement of COL7A1 levels in human samples including serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for a variety of research applications.COL7A1 is a critical component of type VII collagen, which plays a critical role in anchoring fibrils that attach the basement membrane to the underlying dermis. Mutations in the COL7A1 gene can lead to a rare genetic disorder known as dystrophic epidermolysis bullosa (DEB), characterized by blistering and skin fragility.
This ELISA kit provides researchers with valuable insights into COL7A1 levels, aiding in the study of DEB and other related conditions.Whether investigating skin disorders, wound healing, or collagen biology, the Human Collagen Alpha 1 (VII) Chain (COL7A1) ELISA Kit is a reliable and precise tool for your research needs. Trust in its performance to deliver accurate and meaningful results for your studies.
| Product Name: | Human Collagen alpha-1 (VII)chain/COL7A1 ELISA Kit |
| Product Code: | HUFI01350 |
| Size: | 96 Assays |
| Alias: | COL7A1, Collagen alpha-1, VII chain, Long-chain collagen, LC collagen, EBD1, EBDCT, EBR1 |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human COL7A1 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 COL7A1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human COL7A1 in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human COL7A1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
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| 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 | Q02388 |
| UniProt Protein Function: | COL7A1: the alpha chain of type VII collagen, an extra-cellular basement membrane protein restricted to the zone beneath stratified squamous epithelia. Type VII collagen fibrils are composed of three identical alpha collagen chains. Forms anchoring fibrils, which may contribute to epithelial basement membrane organization and adherence by interacting with extracellular matrix (ECM) proteins such as type IV collagen. Dystrophic epidermolysis bullosa can result from defects in this protein or an autoimmune response made to type VII collagen. Homotrimer. Interacts with MIA3, facilitating its loading into transport carriers and subsequent secretion. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Secreted; Extracellular matrix; Secreted, signal peptide; Inhibitor Chromosomal Location of Human Ortholog: 3p21.1 Cellular Component: basement membrane; collagen type VII; endoplasmic reticulum lumen; ER to Golgi transport vesicle; ER-Golgi intermediate compartment membrane; extracellular matrix; extracellular region; extracellular space; Golgi membrane Molecular Function:identical protein binding; protein binding; serine-type endopeptidase inhibitor activity Biological Process: cell adhesion; cellular protein metabolic process; collagen catabolic process; COPII coating of Golgi vesicle; epidermis development; ER to Golgi vesicle-mediated transport; extracellular matrix disassembly; extracellular matrix organization and biogenesis; post-translational protein modification; protein amino acid N-linked glycosylation via asparagine Disease: Epidermolysis Bullosa Dystrophica, Autosomal Dominant; Epidermolysis Bullosa Dystrophica, Autosomal Recessive; Epidermolysis Bullosa Dystrophica, Pretibial; Epidermolysis Bullosa Pruriginosa; Epidermolysis Bullosa With Congenital Localized Absence Of Skin And Deformity Of Nails; Nail Disorder, Nonsyndromic Congenital, 8; Transient Bullous Dermolysis Of The Newborn |
| NCBI Summary: | This gene encodes the alpha chain of type VII collagen. The type VII collagen fibril, composed of three identical alpha collagen chains, is restricted to the basement zone beneath stratified squamous epithelia. It functions as an anchoring fibril between the external epithelia and the underlying stroma. Mutations in this gene are associated with all forms of dystrophic epidermolysis bullosa. In the absence of mutations, however, an acquired form of this disease can result from an autoimmune response made to type VII collagen. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q02388 |
| NCBI GenInfo Identifier: | 1345650 |
| NCBI Gene ID: | 1294 |
| NCBI Accession: | Q02388.2 |
| UniProt Secondary Accession: | Q02388,Q14054, Q16507, |
| UniProt Related Accession: | Q02388 |
| Molecular Weight: | 292,267 Da |
| NCBI Full Name: | Collagen alpha-1(VII) chain |
| NCBI Synonym Full Names: | collagen type VII alpha 1 |
| NCBI Official Symbol: | COL7A1 |
| NCBI Official Synonym Symbols: | EBD1; EBR1; EBDCT; NDNC8 |
| NCBI Protein Information: | collagen alpha-1(VII) chain |
| UniProt Protein Name: | Collagen alpha-1(VII) chain |
| UniProt Synonym Protein Names: | Long-chain collagen; LC collagen |
| UniProt Gene Name: | COL7A1 |
| UniProt Entry Name: | CO7A1_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. |
