The Mouse Disabled Homolog 2 (DAB2) ELISA Kit is a powerful tool designed for the accurate detection of DAB2 levels in mouse serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring precise and reliable results for a variety of research applications.DAB2 is a critical protein involved in cellular signaling pathways and is known to play a role in regulating cell growth, migration, and differentiation. Dysregulation of DAB2 expression has been linked to various diseases, including cancer and cardiovascular disorders, making it an important biomarker for studying these conditions and exploring potential therapeutic interventions.
With its advanced technology and quality performance, the Mouse DAB2 ELISA Kit is an invaluable resource for researchers seeking to deepen their understanding of DAB2 biology and its implications in disease pathogenesis.
Adapter protein that functions as clathrin-associated sorting protein (CLASP) required for clathrin-mediated endocytosis of selected cargo proteins. Can bind and assemble clathrin, and binds simultaneously to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and cargos containing non-phosphorylated NPXY internalization motifs, such as the LDL receptor, to recruit them to clathrin-coated pits. Can function in clathrin-mediated endocytosis independently of the AP-2 complex. Involved in endocytosis of integrin beta-1; this function seems to redundant with the AP-2 complex and seems to require DAB2 binding to endocytosis accessory EH domain-containing proteins such as EPS15, EPS15L1 and ITSN1. Involved in endocytosis of cystic fibrosis transmembrane conductance regulator/CFTR. Isoform p96 is involved in endocytosis of megalin/LRP2 lipoprotein receptor during embryonal development. Required for recycling of the TGF-beta receptor. Isoform p67 is not involved in LDL receptor endocytosis. Involved in CFTR trafficking to the late endosome. Involved in several receptor-mediated signaling pathways. Involved in TGF-beta receptor signaling and facilitates phosphorylation of the signal transducer SMAD2. Mediates TFG-beta-stimulated JNK activation. May inhibit the canoniocal Wnt/beta-catenin signaling pathway by stabilizing the beta-catenin destruction complex through a competing association with axin preventing its dephosphorylation through protein phosphatase 1 (PP1). Sequesters LRP6 towards clathrin-mediated endocytosis, leading to inhibition of Wnt/beta-catenin signaling. May activate non-canonical Wnt signaling. In cell surface growth factor/Ras signaling pathways proposed to inhibit ERK activation by interrupting the binding of GRB2 to SOS1 and to inhibit SRC by preventing its activating phosphorylation at 'Tyr-419'. Proposed to be involved in modulation of androgen receptor (AR) signaling mediated by SRC activation; seems to compete with AR for interaction with SRC. Plays a role in the CSF-1 signal transduction pathway. Plays a role in cellular differentiation. Involved in cell positioning and formation of visceral endoderm (VE) during embryogenesis and proposed to be required in the VE to respond to Nodal signaling coming from the epiblast. Required for the epithelial to mesenchymal transition, a process necessary for proper embryonic development. May be involved in myeloid cell differentiation and can induce macrophage adhesion and spreading. Isoform p67 may be involved in transcriptional regulation. May act as a tumor suppressor.
Uniprot:
P98078
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Disabled homolog 2
Sub Unit:
Interacts (via NPXY motif) with DAB2 (via PID domain). Can interact (via PID domain) with LDLR, APP, APLP1 and APLP2, and weakly with INPP5D (via NPXY motifs); the interaction is impaired by tyrosine phosphorylation of the respective NPXY motifs. Can weakly interact (via PID domain) with LRP1 (via NPXY motif); the interaction is enhanced by tyrosine phosphorylation of the NPXY motif. Interacts with LRP2 (via NPXY motif); the interaction is not affected by tyrosine phosphorylation of the NPXY motif. Interacts with clathrin; in vitro can assemble clathrin triskelia into polyhedral coats. Interacts with AP2A2, ITGB1, ITGB3, ITGB5, PIAS2, DAB2IP, NOSTRIN, FCHO1, DVL3 and EPS15L1. Interacts with SH3KBP1 (via SH3 domains). Interacts with GRB2; competes with SOS1 for binding to GRB2 and the interaction is enhanced by EGF and NT-3 stimulation. Isoform p96 interacts with EPS15 and ITSN1; isoform p67 does not interact with EPS15 and only weakly interacts with ITSN1. Interacts with MAP3K7; the interaction is induced by TGF-beta stimulation and may mediate TGF-beta stimulated JNK activation. Interacts with AXIN1 and PPP1CA; the interactions are mutually exclusive. Interacts with the globular tail of MYO6. Interacts (via DPF motifs) with FCHO2; the interaction is direct and required for DAB2-mediated LDLR endocytosis. Interacts with LRP6; the interaction involves LRP6 phosphorylation by CK2 and sequesters LRP6 towards clathrin-mediated endocytosis. Associates with the TGF-beta receptor complex (Probable). Interacts with SMAD2 and SMAD3; the interactions are enhanced upon TGF-beta stimulation. Interacts with GRB2; the interaction is enhanced by EGF and NT-3 stimulation. Interacts with SRC; the interaction is enhanced by EGF stimulation.
Subcellular Location:
Isoform p67 Cytoplasm Nucleus Diffuse localization in the cytoplasm; does not localize to clathrin-coated pits.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
DAB2: an adaptor protein that interacts with Grb2, myosin VI, SMAD2/3, DIP1/2, Dvl-3, the integrin betasubunit, and c-Src. Negatively regulates canonical Wnt signaling by stabilizing the beta-catenin degradation complex. A component of the CSF-1 signal transduction pathway. Phosphorylated DAB2 inhibits adhesion and integrin signaling. Two alternatively spliced isoforms are described.Protein type: Tumor suppressor; Adaptor/scaffoldCellular Component: focal adhesion; intracellular membrane-bound organelle; clathrin-coated vesicle; clathrin coated vesicle membrane; coated pit; clathrin coat of coated pit; membrane; perinuclear region of cytoplasm; cytoplasm; apical plasma membrane; plasma membrane; nucleolus; cytoplasmic vesicle; nucleusMolecular Function: integrin binding; protein C-terminus binding; phosphatidylinositol-4,5-bisphosphate binding; clathrin binding; protein binding; SMAD binding; phosphoinositide bindingBiological Process: pinocytosis; cellular morphogenesis during differentiation; positive regulation of cell adhesion; apoptosis; multicellular organismal development; positive regulation of transcription, DNA-dependent; positive regulation of endocytosis; excretion; activation of JNK activity; clathrin cage assembly; protein transport; positive regulation of RNA elongation from RNA polymerase II promoter; positive regulation of aldosterone biosynthetic process; transport; cell differentiation; negative regulation of protein binding; positive regulation of receptor recycling; negative regulation of epithelial cell proliferation; receptor-mediated endocytosis; Wnt receptor signaling pathway; leading edge cell differentiation; in utero embryonic development; positive regulation of transforming growth factor beta receptor signaling pathway; endocytosis; response to steroid hormone stimulus; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; negative regulation of cell growth; positive regulation of protein amino acid phosphorylation; negative regulation of transcription, DNA-dependent; positive regulation of cell migration; endoderm development; negative regulation of apoptosis
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.
