Description
Rat ITGa6 ELISA Kit (Small Sample Volume)
Rat ITGa6 ELISA kits are used to detect Integrin Alpha 6 (ITGa6) in tissue homogenates and other biological samples. ITGa6, also known as CD49f, is an integrin alpha subunit that pairs with either beta-1 or beta-4 to form laminin receptors. The alpha-6 beta-1 integrin is present on platelets and on oocytes, where it takes part in sperm-egg fusion, while alpha-6 beta-4 is found in epithelial cells and plays a critical structural role in the hemidesmosome that anchors epithelium to the basement membrane. Alpha-6 beta-4 also binds NRG1, IGF1 and IGF2, and these interactions are needed for ERBB and IGF signalling. ITGa6 is widely used as a marker of stem and progenitor cells in several tissues and is frequently upregulated in carcinomas. Loss-of-function variants cause junctional epidermolysis bullosa with pyloric atresia, a severe blistering skin disorder.
| Product Name: | Rat ITGa6 (Integrin Alpha 6) ELISA (Small Sample Volume) |
| SKU: | AEKE10041 |
| Size: | 96 Assays |
| Reactivity: | Rat |
| Synonyms: | CD49f, CD49-F, ITG-A6, ITGA6B, VLA-6, CD49 antigen-like family member F, Processed integrin alpha-6 |
| Detection Method: | Sandwich |
| Sensitivity: | 0.116 ng/mL |
| Range: | 0.32-20 ng/mL |
| Standard: | 20 ng/mL |
| Assay Length: | 3.5h |
| Sample Type: | Tissue Homogenates And Other Biological Fluids. |
The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Rat ITGa6. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to Rat ITGa6. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Rat ITGa6, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Rat ITGa6 in the samples is then determined by comparing the OD of the samples to the standard curve.
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*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.
| Step | Protocol |
|---|---|
| 1. | After the kit is equilibrated at room temperature, add µL of Standard Working Buffer (gradually diluted according to the instructions) or µL of sample to each well, and incubate at 37°C for 80 minutes. |
| 2. | Discard the liquid in the plate, add µL 1× Wash Buffer to each well, and wash the plate 3 times. After pat it dry against clean absorbent paper, add µL Biotinylated Antibody Working Solution (1×) to each well, incubate at 37°C for 50 minutes. |
| 3. | Discard the liquid in the plate, add µL 1× Wash Buffer to each well, and wash the plate 3 times. After pat it dry against clean absorbent paper, add µL 1× Streptavidin-HRP Working Solution to each well, incubate at 37°C for 50 minutes. |
| 4. | Discard the liquid in the plate, add µL 1× Wash Buffer to each well, and wash the plate 5 times. After pat it dry against clean absorbent paper, add 90 µL TMB Substrate Solution to each well, incubate at 37°C for 20 minutes in the dark. |
| 5. | Add 50 µL Stop Solution to each well, shake plate on a plate shaker for 1 minute to mix. Record the OD at 450 nm immediately, calculation of the results. |
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 | Samples should be collected into a serum separator tube. After clotting for 2 hours at room temperature or overnight at 4°C, and then centrifuging at 1000 × g for 20 minutes. Assay freshly prepared serum immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 1000 × g and 2-8°C for 15 minutes within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles. |
| Tissue homogenates | 1. Rinse the tissues in pre-cooled PBS to completely remove excess blood, and weigh them before homogenization. 2. Mince the tissues and homogenize in fresh lysis buffer (PBS for most tissues). Use a glass homogenizer on ice. 3. Ultrasound the suspension until the solution is clear. 4. Centrifuge for 5 minutes at 10000 × g, collect the supernatant and assay immediately or store at ≤ -20°C. |
| Cell lysates | 1. Wash adherent cells with PBS, detach with trypsin, and centrifuge at 1000 × g for 5 minutes. 2. Wash cells 3 times in PBS. 3. Resuspend cells in fresh lysis buffer at 107 cells/mL. Ultrasound if necessary. 4. Centrifuge at 1500 × g for 10 minutes at 2-8°C to remove debris. Assay immediately or store at ≤ -20°C. |
| Urine | Collect mid-stream first urine of the day directly into a sterile container. Centrifuge to remove particulate matter. Assay immediately or aliquot and store at ≤ -20°C. Avoid repeated freeze-thaw cycles. |
| Saliva | Collect saliva using a collection device. Centrifuge at 1000 × g for 15 minutes at 2-8°C. Remove particulates and assay immediately or aliquot and store at ≤ -20°C. Avoid repeated freeze-thaw cycles. |
| Feces | Dry feces weighing more than 50 mg were collected. Wash with PBS (w:v = 1:9). Sonicate and centrifuge at 5000 × g for 10 minutes. Collect the supernatant and assay immediately. |
| CSF (Cerebrospinal fluid) | Remove particulates by centrifugation. Assay immediately or aliquot and store at ≤ -20°C. Avoid repeated freeze-thaw cycles. |
| Cell culture supernatant | Centrifuge samples at 1000 × g for 20 minutes. Collect the supernatant and assay immediately or store at -20°C or -80°C. Avoid repeated freeze-thaw cycles. |
| Uniprot ID: | - |
| Research Area: | CD & Adhesion molecule |
