Description
| Product Name: | Rat P2X purinoceptor 7 (P2rx7) ELISA Kit |
| Product Code: | RTEB0783 |
| Alias: | P2X purinoceptor 7, P2X7, ATP receptor, P2Z receptor, Purinergic receptor, P2rx7 |
| Uniprot: | Q64663 |
| Reactivity: | Rat |
| Range: | Please contact us for more information |
| Detection Method: | Sandwich |
| Size: | 96 Assay |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | P2X7: a receptor for ATP that acts as a ligand-gated ion channel. Responsible for ATP-dependent lysis by macrophages through the formation of membrane pores permeable to large molecules. Activation of this nuclear receptor by ATP in the cytoplasm may be a mechanism by which cellular activity can be coupled to changes in gene expression. Alternatively spliced variants encoding different isoforms have been identified. Could function in both fast synaptic transmission and the ATP-mediated lysis of antigen-presenting cells. Phosphorylation results in its inactivation.Protein type: Membrane protein, integral; Membrane protein, multi-pass; Channel, ligand-gated; Receptor, misc.Cellular Component: neuron projection; protein complex; integral to plasma membrane; terminal button; integral to nuclear inner membrane; intercellular junction; cytosol; lipid raft; membrane; cell soma; cytoplasm; plasma membrane; synapse; intracellular; neuromuscular junction; external side of plasma membraneMolecular Function: protein serine/threonine kinase activator activity; protein binding; copper ion binding; zinc ion binding; channel activity; lipopolysaccharide binding; magnesium ion binding; receptor activity; ATP binding; purinergic nucleotide receptor activity; receptor binding; ATP-gated cation channel activityBiological Process: positive regulation of catalytic activity; positive regulation of apoptosis; positive regulation of T cell mediated cytotoxicity; activation of MAPK activity; cytolysis; programmed cell death; positive regulation of glutamate secretion; response to lipopolysaccharide; positive regulation of interleukin-1 beta secretion; sensory perception of pain; response to organic cyclic substance; protein amino acid phosphorylation; positive regulation of interleukin-1 alpha secretion; cell surface receptor linked signal transduction; multicellular organismal protein catabolic process; ceramide biosynthetic process; positive regulation of cytolysis; T cell homeostasis; response to electrical stimulus; positive regulation of gamma-aminobutyric acid secretion; response to drug; mitochondrion organization and biogenesis; collagen metabolic process; positive regulation of interleukin-6 production; positive regulation of cytoskeleton organization and biogenesis; regulation of sodium ion transport; protein oligomerization; phospholipid translocation; phagolysosome formation; membrane depolarization; defense response to Gram-positive bacterium; positive regulation of protein secretion; NAD transport; response to bacterium; response to mechanical stimulus; response to zinc ion; cell volume homeostasis; response to calcium ion; phospholipid transfer to membrane; cell morphogenesis; generation of action potential; response to organic substance; T cell proliferation; organic cation transport; positive regulation of mitochondrial depolarization; positive regulation of MAPKKK cascade; plasma membrane organization and biogenesis; calcium ion transport; pore complex biogenesis; inflammatory response; negative regulation of bone resorption; cation transport; negative regulation of MAPKKK cascade; membrane protein ectodomain proteolysis; bleb formation; positive regulation of bone mineralization; response to ATP; positive regulation of ossification; positive regulation of interleukin-1 beta production; synaptic vesicle exocytosis; homeostasis of number of cells within a tissue; skeletal morphogenesis; release of sequestered calcium ion into cytosol; positive regulation of prostaglandin secretion; cellular response to extracellular stimulus; protein processing; gene expression; positive regulation of protein amino acid phosphorylation; positive regulation of cytokine secretion; membrane budding |
| UniProt Protein Details: | |
| NCBI Summary: | an ATP receptor; may be involved in fast synaptic transmission and the ATP-mediated lysis of antigen-presenting cells [RGD, Feb 2006] |
| UniProt Code: | Q64663 |
| NCBI GenInfo Identifier: | 2499425 |
| NCBI Gene ID: | 29665 |
| NCBI Accession: | Q64663.1 |
| UniProt Secondary Accession: | Q64663 |
| UniProt Related Accession: | Q64663 |
| Molecular Weight: | 68,392 Da |
| NCBI Full Name: | P2X purinoceptor 7 |
| NCBI Synonym Full Names: | purinergic receptor P2X, ligand-gated ion channel, 7 |
| NCBI Official Symbol: | P2rx7 |
| NCBI Official Synonym Symbols: | |
| NCBI Protein Information: | P2X purinoceptor 7; ATP receptor; P2Z receptor; P2X7 purinoceptor; purinergic receptor P2X7 |
| UniProt Protein Name: | P2X purinoceptor 7 |
| UniProt Synonym Protein Names: | ATP receptor; P2Z receptor; Purinergic receptor |
| Protein Family: | P2X purinoceptor |
| UniProt Gene Name: | P2rx7 |
| UniProt Entry Name: | P2RX7_RAT |
| 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. |
