The Mouse TIRAP (Toll/interleukin-1 receptor domain-containing adapter protein) ELISA Kit is expertly designed for the precise measurement of TIRAP levels in mouse serum, plasma, and cell culture supernatants. With its superior sensitivity and specificity, this kit delivers accurate and reproducible results, making it the perfect choice for a wide array of research purposes.TIRAP is a critical adaptor protein involved in the signaling pathways of the immune system, specifically in response to Toll-like receptors and interleukin-1 receptors.
Its role in regulating inflammatory responses and immune cell activation makes it a key player in various diseases, including infectious diseases, autoimmune disorders, and inflammatory conditions. The Mouse TIRAP ELISA Kit provides researchers with a valuable tool to further investigate the role of TIRAP in these diseases and explore potential therapeutic interventions.
Product Name:
Mouse Tirap ELISA Kit
SKU:
MOEB2192
Size:
96T
Target:
Mouse Tirap
Synonyms:
Adaptor protein Wyatt, MyD88 adapter-like protein, TIR domain-containing adapter protein, Mal
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Mouse
Detection Range:
31.2-2000pg/mL
Sensitivity:
15.7pg/mL
Intra CV:
5.2%
Inter CV:
6.8%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
96-105%
99-109%
93-103%
97-107%
EDTA Plasma(N=5)
103-115%
105-114%
97-105%
87-97%
Heparin Plasma(N=5)
101-111%
99-109%
102-114%
108-117%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
98
92-104
Plasma
100
94-106
Function:
Adapter involved in the TLR2 and TLR4 signaling pathways in the innate immune response. Acts via IRAK2 and TRAF-6, leading to the activation of NF-kappa-B, MAPK1, MAPK3 and JNK, and resulting in cytokine secretion and the inflammatory response (By similarity). Positively regulates the production of TNF-alpha and interleukin-6.
Uniprot:
Q99JY1
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Toll/interleukin-1 receptor domain-containing adapter protein
Sub Unit:
Homodimer. Also forms heterodimers with MYD88. Interacts with BMX and TBK1 (By similarity). Interacts with TLR4 and IRAK2 via their respective TIR domains. Interacts with EIF2AK2. Does not interact with IRAK1-1, nor TLR9. May interact with PIK3AP1.
Research Area:
Immunology
Subcellular Location:
Cytoplasm Cell membrane Membrane Colocalizes with DAB2IP at the plasma membrane.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
TIRAP: an adapter protein involved in the TLR4 signaling pathway in the innate immune response. Acts via IRAK2 and TRAF-6, leading to the activation of NF-kappa-B, ERK1, ERK2 and JNK, resulting in cytokine secretion and the inflammatory response. Homodimerizes. Also forms heterodimers with MyD88. Binds to TLR4 and IRAK2 via their respective TIR domains. Binds to PKR and TBK1. Does not interact with IRAK1, nor TLR9. Genetic variations in TIRAP can influence susceptibility or resistance to infectious diseases, including invasive pneumococcal disease, malaria and tuberculosis. Three isoforms of the human protein are produced by alternative splicing.Protein type: Adaptor/scaffoldCellular Component: nucleoplasm; intercellular bridge; membrane; endocytic vesicle; cytoplasm; plasma membrane; peroxisome; intracellularMolecular Function: protein binding; phosphatidylinositol-4,5-bisphosphate binding; protein homodimerization activity; protein kinase C binding; protein heterodimerization activityBiological Process: I-kappaB kinase/NF-kappaB cascade; positive regulation of interleukin-12 production; positive regulation of interleukin-6 biosynthetic process; positive regulation of JNK cascade; response to lipopolysaccharide; signal transduction; activation of JNK activity; activation of NF-kappaB transcription factor; positive regulation of interleukin-8 production; toll-like receptor 5 signaling pathway; cell surface receptor linked signal transduction; positive regulation of interleukin-15 production; positive regulation of innate immune response; positive regulation of B cell proliferation; inflammatory response; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of toll-like receptor 3 signaling pathway; immune system process; positive regulation of toll-like receptor 4 signaling pathway; positive regulation of tumor necrosis factor production; regulation of interleukin-15 production; positive regulation of toll-like receptor 2 signaling pathway; regulation of innate immune response; defense response to Gram-positive bacterium; response to bacterium; innate immune response; myeloid cell differentiation; regulation of interferon-beta production
toll/interleukin-1 receptor domain-containing adapter protein
NCBI Synonym Full Names:
toll-interleukin 1 receptor (TIR) domain-containing adaptor protein
NCBI Official Symbol:
Tirap
NCBI Official Synonym Symbols:
Mal; Wyatt; Tlr4ap; AA407980; C130027E04Rik
NCBI Protein Information:
toll/interleukin-1 receptor domain-containing adapter protein
UniProt Protein Name:
Toll/interleukin-1 receptor domain-containing adapter protein
UniProt Synonym Protein Names:
Adaptor protein Wyatt; MyD88 adapter-like protein
Protein Family:
Toll/interleukin-1 receptor domain-containing adapter protein
UniProt Gene Name:
Tirap
UniProt Entry Name:
TIRAP_MOUSE
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.
