Rat Axin-1 (Axin1) ELISA Kit (RTEB1683)- High Sensitivity

Product Name:	Rat Axin-1 (Axin1) ELISA Kit
SKU:	RTEB1683
Size:	96T
Target:	Rat Axin-1 (Axin1)
Synonyms:	Axis inhibition protein 1, rAxin, Axin
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.156-10ng/ml
Sensitivity:	0.078ng/mL

Intra CV:

4.3%

Inter CV:

9.5%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	104-114%	108-118%	107-117%	108-120%
EDTA Plasma(N=5)	104-113%	91-101%	113-122%	87-99%
Heparin Plasma(N=5)	81-93%	92-101%	99-108%	96-105%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	95	89-101
Plasma	97	91-103

Function:

Component of the beta-catenin destruction complex required for regulating CTNNB1 levels through phosphorylation and ubiquitination, and modulating Wnt-signaling. Controls dorsoventral patterning via two opposing effects; down-regulates beta-catenin to inhibit the Wnt signaling pathway and ventralize embryos, but also dorsalizes embryos by activating a Wnt-independent JNK signaling pathway. Also facilitates the phosphorylation of APC by GSK3B. Facilitates the phosphorylation of TP53 by HIPK2 upon ultraviolet irradiation. Enhances TGF-beta signaling by recruiting the RNF111 E3 ubiquitin ligase and promoting the degradation of inhibitory SMAD7.

Uniprot:	O70239
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Axin-1
Sub Unit:	Homodimer (By similarity). Interacts with ZBED3; the interaction is direct, enhanced by protein kinase GSK3B and casein kinase CSNK1E activities and decreases GSK3B-induced beta-catenin serine and threonine phosphorylations (By similarity). Component of the AXIN1-HIPK2-TP53 complex (By similarity). Interacts directly in the complex with TP53 and HIPK2. Interacts with DAXX; the interaction stimulates the interaction of DAXX with TP53, stimulates 'Ser-46' phosphorylation of TP53 and induces cell death on UV irradiation. Also binds ANKRD6, PIAS1, PIAS2, PIAS4, MAP3K1, MAP3K4, SUMO1, SMAD6, SMAD7 and RNF111. Interacts with DIXDC1; the interaction prevents interaction with MAP3K1. Interacts with MDFI; the interaction decreases AXIN1-mediated JUN N-terminal kinase (JNK) activation. Interacts with MDFIC; the interaction inhibits beta-cateninin-mediated signaling and AXIN1-mediated JUN N-terminal kinase (JNK) activation. Interacts with LRP5 (via its phosphorylated PPPSP motifs); the interaction is stimulated by WNT1 and GSK3B and activates beta-catenin signaling. Interacts (via the C-terminal) with PPP1CA; the interaction dephosphorylates AXIN1 and regulates interaction with GSK3B. Interacts with PPP2CA; the interaction dephosphorylates AXIN1 (By similarity). Component of the beta-catenin destruction complex, containing at least, CTNNB1, an axin and GSK3B, that regulates CTNNB1 protein levels through phosphorylation and ubiquitination. Interacts with CTNNB1 (via the armadillo repeats 2-7). Interacts with GSK3B; the interaction hyperphosphorylates CTNNB1 leading to its ubiquitination and destruction. Interacts with MACF1. Found in a complex composed of MACF1, APC, AXIN1, CTNNB1 and GSK3B. Interacts with TNKS. Interacts with DAB2; the interaction is mutually exclusive with the AXIN1:PPP1CA interaction.
Research Area:	Cancer
Subcellular Location:	Cytoplasm Nucleus Cell membrane Membrane On UV irradiation, translocates to the nucleus and colocalizes with DAAX (By similarity). MACF1 is required for its translocation to cell membrane (By similarity).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	axin 1: is a negative regulator of the Wnt pathway, which is critical in stem cell signaling, morphogenesis, the mesenchymal-epithelial transition, and many cancers. Axin-1 functions as a tumor suppressor. Probably facilitates the phosphorylation of beta-catenin and APC by GSK3B, leading to their ubiquitination and subsequent proteolysis. Wild-type axin 1 can induce apoptosis in hepatocellular and colorectal cancer cells. Is downregulated during progression of esophageal squamous cell carcinoma. Mutation of the axin-1 gene is associated with hepatocellular carcinoma, anaplastic thyroid cancer, medulloblastoma and colorectal cancer. May have a role in oncogenesis in Hodgkin lymphoma. Axin1/2 mediate cross-talk between TGF-beta and Wnt signaling pathways.Protein type: Adaptor/scaffold; Tumor suppressorChromosomal Location of Human Ortholog: 10q12Cellular Component: beta-catenin destruction complex; cell cortex; cytoplasm; cytoplasmic microtubule; cytoplasmic vesicle; Golgi apparatus; lateral plasma membrane; nucleus; perinuclear region of cytoplasm; plasma membrane; postsynaptic density; protein complexMolecular Function: beta-catenin binding; enzyme binding; GTPase activator activity; identical protein binding; p53 binding; protein binding; protein C-terminus binding; protein complex scaffold activity; protein domain specific binding; protein homodimerization activity; protein kinase binding; protein self-association; receptor binding; receptor signaling complex scaffold activity; signal transducer activity; SMAD binding; ubiquitin protein ligase bindingBiological Process: activation of JNK activity; activation of protein kinase activity; adult walking behavior; apoptosis; axial mesoderm development; axial mesoderm formation; cellular protein complex assembly; cytoplasmic microtubule organization; determination of left/right symmetry; dorsal/ventral axis specification; dorsal/ventral pattern formation; embryonic eye morphogenesis; erythrocyte homeostasis; forebrain anterior/posterior pattern specification; hemoglobin metabolic process; in utero embryonic development; muscle cell development; negative regulation of fat cell differentiation; negative regulation of protein metabolic process; negative regulation of Wnt signaling pathway; neurological system process; nucleocytoplasmic transport; olfactory placode formation; optic placode formation; positive regulation of GTPase activity; positive regulation of JNK cascade; positive regulation of JUN kinase activity; positive regulation of peptidyl-serine phosphorylation; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; positive regulation of protein catabolic process; positive regulation of protein kinase activity; positive regulation of protein phosphorylation; positive regulation of protein ubiquitination; positive regulation of transcription, DNA-templated; positive regulation of transforming growth factor beta receptor signaling pathway; positive regulation of ubiquitin-protein transferase activity; protein catabolic process; protein homooligomerization; protein polyubiquitination; regulation of protein amino acid phosphorylation; sensory perception of sound; Wnt receptor signaling pathway involved in forebrain neuron fate commitment; Wnt receptor signaling pathway through beta-catenin; Wnt signaling pathway
UniProt Protein Details:
NCBI Summary:
UniProt Code:	O70239
NCBI GenInfo Identifier:	13242326
NCBI Gene ID:	79257
NCBI Accession:	NP_077381.1
UniProt Secondary Accession:	O70239
UniProt Related Accession:	O70239
Molecular Weight:	92,285 Da
NCBI Full Name:	axin-1
NCBI Synonym Full Names:	axin 1
NCBI Official Symbol:	Axin1
NCBI Official Synonym Symbols:	Axin; axin-1
NCBI Protein Information:	axin-1
UniProt Protein Name:	Axin-1
UniProt Synonym Protein Names:	Axis inhibition protein 1; rAxin
Protein Family:
UniProt Gene Name:	Axin1
UniProt Entry Name:

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.