Description
Rat NF-kappa-B inhibitor alpha (Nfkbia) ELISA Kit
The Rat NF-kappa B Inhibitor Alpha (NFKBIA) ELISA Kit is a powerful tool for detecting and measuring NFKBIA levels in rat samples, including serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for various research applications.NFKBIA is a key regulator of the NF-kappa B signaling pathway, playing a critical role in the immune response, inflammation, and cell survival. Dysregulation of NFKBIA has been linked to various diseases, including autoimmune disorders, inflammatory conditions, and cancer.
By accurately measuring NFKBIA levels, researchers can gain valuable insights into these disease mechanisms and potential therapeutic targets.Overall, the Rat NF-kappa B Inhibitor Alpha (NFKBIA) ELISA Kit is a valuable tool for studying the NF-kappa B pathway and its role in disease pathogenesis, offering researchers a reliable and efficient way to measure NFKBIA levels in rat samples.
| Product Name: | Rat NF-kappa-B inhibitor alpha (Nfkbia) ELISA Kit |
| SKU: | RTEB1234 |
| Size: | 96T |
| Target: | Rat NF-kappa-B inhibitor alpha (Nfkbia) |
| Synonyms: | I-kappa-B-alpha, RL/IF-1, IkB-alpha, Ikba |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Rat |
| Detection Range: | 0.156-10ng/mL |
| Sensitivity: | 0.078ng/mL |
| Intra CV: | 4.6% | ||||||||||||||||||||
| Inter CV: | 8.2% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Inhibits the activity of dimeric NF-kappa-B/REL complexes by trapping REL dimers in the cytoplasm through masking of their nuclear localization signals. On cellular stimulation by immune and proinflammatory responses, becomes phosphorylated promoting ubiquitination and degradation, enabling the dimeric RELA to translocate to the nucleus and activate transcription. |
| Uniprot: | Q63746 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant rat NF-kappa-B inhibitor alpha |
| Sub Unit: | Interacts with RELA; the interaction requires the nuclear import signal. Interacts with NKIRAS1 and NKIRAS2. Part of a 70-90 kDa complex at least consisting of CHUK, IKBKB, NFKBIA, RELA, IKBKAP and MAP3K14. Interacts with RWDD3; the interaction enhances sumoylation. Interacts (when phosphorylated at the 2 serine residues in the destruction motif D-S-G-X(2,3,4)-S) with BTRC. Associates with the SCF(BTRC) complex, composed of SKP1, CUL1 and BTRC; the association is mediated via interaction with BTRC. Part of a SCF(BTRC)-like complex lacking CUL1, which is associated with RELA; RELA interacts directly with NFKBIA. Interacts with PRMT2. Interacts with PRKACA in platelets; this interaction is disrupted by thrombin and collagen (By similarity). Interacts with HIF1AN (By similarity). Interacts with MEFV (By similarity). Interacts with DDRGK1; positively regulates NFKBIA phosphorylation and degradation. |
| Research Area: | Immunology |
| Subcellular Location: | Cytoplasm Nucleus Shuttles between the nucleus and the cytoplasm by a nuclear localization signal (NLS) and a CRM1-dependent nuclear export. |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | IkB-alpha: a regulatory protein that inhibits NF-kappa-B by complexing with and trapping it in the cytoplasm. May be involved in regulation of transcriptional responses to NF-kappa-B, including cell adhesion, immune and proinflammatory responses, apoptosis, differentiation and growth. Controlled by sequential serine-phosphorylation, ubiquitination and degradation. |
| UniProt Protein Details: | Protein type:Inhibitor; DNA-binding Cellular Component: cytoplasm; cytosol; nucleus; plasma membrane; protein complex Molecular Function:enzyme binding; heat shock protein binding; identical protein binding; NF-kappaB binding; nuclear localization sequence binding; protein complex binding; ubiquitin protein ligase binding Biological Process: cytoplasmic sequestering of NF-kappaB; cytoplasmic sequestering of transcription factor; inhibition of NF-kappaB transcription factor; lipopolysaccharide-mediated signaling pathway; negative regulation of myeloid cell differentiation; negative regulation of Notch signaling pathway; positive regulation of cellular protein metabolic process; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; protein import into nucleus, translocation; regulation of cell proliferation; regulation of gene expression; response to exogenous dsRNA; response to lipopolysaccharide; response to muramyl dipeptide; toll-like receptor 4 signaling pathway |
| NCBI Summary: | inhibitor of NF-kappa-B; binds NF kappa B and retains it in the cytoplasm [RGD, Feb 2006] |
| UniProt Code: | Q63746 |
| NCBI GenInfo Identifier: | 160333919 |
| NCBI Gene ID: | 25493 |
| NCBI Accession: | NP_001099190.2 |
| UniProt Secondary Accession: | Q63746,B2RYS5, |
| UniProt Related Accession: | Q63746 |
| Molecular Weight: | 35,017 Da |
| NCBI Full Name: | NF-kappa-B inhibitor alpha |
| NCBI Synonym Full Names: | nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha |
| NCBI Official Symbol: | Nfkbia |
| NCBI Official Synonym Symbols: | RL/IF-1 |
| NCBI Protein Information: | NF-kappa-B inhibitor alpha |
| UniProt Protein Name: | NF-kappa-B inhibitor alpha |
| UniProt Synonym Protein Names: | I-kappa-B-alpha; IkB-alpha; IkappaBalpha; RL/IF-1 |
| Protein Family: | NF-kappa-B inhibitor |
| UniProt Gene Name: | Nfkbia |
| UniProt Entry Name: | IKBA_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. |
