Rat Cell Biology ELISA Kits 2
Rat Adenomatous polyposis coli protein (Apc) ELISA Kit
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- SKU:
- RTEB0649
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P70478
- ELISA Type:
- Sandwich
- Reactivity:
- Rat
Description
| Product Name: | Rat Adenomatous polyposis coli protein (Apc) ELISA Kit |
| Product Code: | RTEB0649 |
| Alias: | Adenomatous polyposis coli protein, Protein APC, Apc |
| Uniprot: | P70478 |
| 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: | APC: a tumor suppressor. Defects in APC are a cause of familial adenomatous polyposis (FAP). Promotes rapid degradation of CTNNB1 and participates in Wnt signaling. Activity is correlated with its phosphorylation state. Two splice-variant isoforms have been described.Protein type: Motility/polarity/chemotaxis; Tumor suppressorCellular Component: microtubule; centrosome; neuron projection; nuclear membrane; protein complex; tight junction; cell; dendrite; cell cortex; beta-catenin destruction complex; kinetochore; cell projection; growth cone; cell-cell adherens junction; cell soma; cytoplasmic microtubule; axon; cell projection membrane; lamellipodium; cytoplasm; plasma membrane; synapse; nucleus; lateral plasma membraneMolecular Function: microtubule plus-end binding; protein binding; protease binding; cadherin binding; gamma-catenin binding; microtubule binding; protein complex binding; beta-catenin binding; protein kinase regulator activity; protein kinase bindingBiological Process: skin development; positive regulation of cell adhesion; negative regulation of Wnt receptor signaling pathway; positive regulation of apoptosis; somatic stem cell maintenance; regulation of cell cycle; T cell differentiation in the thymus; Wnt receptor signaling pathway through beta-catenin; regulation of cell migration; cytoplasmic microtubule organization and biogenesis; anterior/posterior pattern formation; muscle maintenance; cytokinesis after mitosis; hair follicle development; positive regulation of epithelial cell differentiation; protein complex assembly; kidney development; cell cycle arrest; neurite development; response to drug; regulation of microtubule-based process; negative regulation of odontogenesis; regulation of osteoblast differentiation; negative regulation of cyclin-dependent protein kinase activity; retina development in camera-type eye; positive regulation of cell division; regulation of epithelial cell differentiation; positive regulation of protein catabolic process; regulation of nitrogen compound metabolic process; positive regulation of cell differentiation; mitotic metaphase/anaphase transition; negative regulation of apoptosis; positive regulation of microtubule polymerization; establishment and/or maintenance of cell polarity; chromosome organization and biogenesis; negative regulation of cell proliferation; regulation of osteoclast differentiation; regulation of cell differentiation; pancreas development; mitotic cell cycle spindle assembly checkpoint; proximal/distal pattern formation; negative regulation of epithelial cell proliferation; cell migration; regulation of attachment of spindle microtubules to kinetochore; Wnt receptor signaling pathway; thymus development; negative regulation of microtubule depolymerization; negative regulation of MAPKKK cascade; axis specification; stem cell maintenance; pattern specification process; positive regulation of pseudopodium formation; dorsal/ventral pattern formation; axonogenesis; response to DNA damage stimulus; positive regulation of cell migration |
| UniProt Protein Details: | |
| NCBI Summary: | binds microtubules; may play a role in the regulation of cell polarity [RGD, Feb 2006] |
| UniProt Code: | P70478 |
| NCBI GenInfo Identifier: | 6978509 |
| NCBI Gene ID: | 24205 |
| NCBI Accession: | NP_036631.1 |
| UniProt Secondary Accession: | P70478 |
| UniProt Related Accession: | P70478 |
| Molecular Weight: | 310,533 Da |
| NCBI Full Name: | adenomatous polyposis coli protein |
| NCBI Synonym Full Names: | adenomatous polyposis coli |
| NCBI Official Symbol: | Apc |
| NCBI Official Synonym Symbols: | RATAPC |
| NCBI Protein Information: | adenomatous polyposis coli protein |
| UniProt Protein Name: | Adenomatous polyposis coli protein |
| UniProt Synonym Protein Names: | |
| Protein Family: | APC-related protein |
| UniProt Gene Name: | Apc |
| UniProt Entry Name: | APC_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. |
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