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Mouse LRP-6 / Low-density lipoprotein receptor-related protein 6 ELISA Kit

SKU:
MOFI00317
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O88572
Sensitivity:
0.375ng/ml
Range:
0.625-40ng/ml
ELISA Type:
Sandwich
Synonyms:
Lrp6, Low-density lipoprotein receptor-related protein 6, LRP-6
Reactivity:
Mouse
Research Area:
Cell Biology
€649
Frequently bought together:

Description

system_update_altDatasheetsystem_update_altMSDS
Product Name:Mouse LRP-6 / Low-density lipoprotein receptor-related protein 6 ELISA Kit
Product Code:MOFI00317
Size:96 Assays
Alias:Lrp6, Low-density lipoprotein receptor-related protein 6, LRP-6
Detection Method:Sandwich ELISA
Application:This immunoassay kit allows for the in vitro quantitative determination of Mouse Lrp6 concentrations in serum plasma and other biological fluids.
Sensitivity:0.375ng/ml
Range:0.625-40ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery: Matrices listed below were spiked with certain level of Mouse Lrp6 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse Lrp6 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)88-10397
EDTA plasma(n=5)85-10596
UFH plasma(n=5)91-10396
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse Lrp6 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)94-105%87-105%85-103%
EDTA plasma(n=5)82-99%86-99%84-101%
UFH plasma(n=5)80-98%81-96%84-94%
Intra Assay:CV <8%
Inter Assay:CV <10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8-12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

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
UniprotO88572
UniProt Protein Function:LRP6: Component of the Wnt-Fzd-LRP5-LRP6 complex that triggers beta-catenin signaling through inducing aggregation of receptor- ligand complexes into ribosome-sized signalsomes. Cell-surface coreceptor of Wnt/beta-catenin signaling, which plays a pivotal role in bone formation. The Wnt-induced Fzd/LRP6 coreceptor complex recruits DVL1 polymers to the plasma membrane which, in turn, recruits the AXIN1/GSK3B-complex to the cell surface promoting the formation of signalsomes and inhibiting AXIN1/GSK3- mediated phosphorylation and destruction of beta-catenin. Required for posterior patterning of the epiblast during gastrulation. Homodimer; disulfide-linked. Forms phosphorylated oligomer aggregates on Wnt-signaling. Forms a WNT-signaling complex formed of a WNT protein, a FZD protein and LRP5 or LRP6. Interacts (via the extracellular domain) with WNT1; the interaction is enhanced by prior formation of the Wnt/Fzd complex. Interacts (via the beta-propeller regions 3 and 4) with WNT3A. Interacts (via the beta-propeller regions 1 and 2) with WNT9B. Interacts with FZD5; the interaction forms a coreceptor complex for Wnt signaling and is inhibited by DKK1 and C1orf187. Interacts (via beta propeller region) with DKK1; the interaction inhibits FZD5/LRP6 complex formation. Interacts with DKK2. Interacts with C1orf187/DRAXIN; the interaction inhibits Wnt signaling. Interacts (via the phosphorylated PPPSP motifs) with AXIN1; the interaction recruits the AXIN1/GSK3B complex to cell surface LRP6 signalsomes. Interacts with GRB10; the interaction prevents AXIN1 binding, thus negatively regulating the Wnt signaling pathway. Interacts (via the extracellular domain) with RSPO1; the interaction activates Wnt/beta-catenin signaling. Interacts (via the extracellular domain) with RSPO3 (via the cysteine rich domain); the interaction activates Wnt/beta-catenin signaling. Interacts (via the beta- propeller regions 1 and 2) with SOST; the interaction competes with DKK1 for binding for inhibiting beta-catenin signaling. Interacts with MESD; the interaction prevents the formation of LRP6 aggregates and targets LRP6 to the plasma membrane. Interacts (via the cytoplasmic domain) with CSNKIE; the interaction phosphorylates LRP6, binds AXIN1 and inhibits AXIN1/GSK3B-mediated phosphorylation of beta-catenin. Interacts with MACF1. Decreased levels on WNT3A stimulation. Widely co-expressed with LRP5 during embryogenesis and in adult tissues. Belongs to the LDLR family.
UniProt Protein Details:

Protein type:Receptor, misc.; Membrane protein, integral

Chromosomal Location of Human Ortholog: 12p13.2

Cellular Component: Golgi apparatus; cell surface; endoplasmic reticulum; early endosome; extracellular region; integral to membrane; caveola; cell soma; early endosome membrane; plasma membrane; synapse; cytoplasmic vesicle; receptor complex

Molecular Function:low-density lipoprotein receptor activity; identical protein binding; Wnt-protein binding; Wnt receptor activity; protein binding; protein homodimerization activity; kinase inhibitor activity; frizzled binding; toxin transporter activity; apolipoprotein binding; receptor binding

Biological Process: radial glial cell differentiation in the forebrain; regulation of fat cell differentiation; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; cerebellum morphogenesis; Wnt receptor signaling pathway through beta-catenin; embryonic pattern specification; bone remodeling; Wnt receptor signaling pathway in forebrain neuroblast division; anterior/posterior pattern formation; cell-cell adhesion; elevation of cytosolic calcium ion concentration; response to folic acid; convergent extension; positive regulation of mesenchymal cell proliferation; midbrain development; embryonic limb morphogenesis; external genitalia morphogenesis; neural crest formation; positive regulation of cell cycle; negative regulation of fat cell differentiation; midbrain-hindbrain boundary development; negative regulation of protein kinase activity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription factor activity; cerebral cortex development; embryonic digit morphogenesis; regulation of ossification; embryonic forelimb morphogenesis; response to peptide hormone stimulus; thalamus development; palate development; embryonic hindlimb morphogenesis; synaptic transmission; negative regulation of protein amino acid phosphorylation; positive regulation of bone resorption; heart looping; neural crest cell differentiation; cell migration involved in gastrulation; negative regulation of epithelial cell proliferation; Wnt receptor signaling pathway; gastrulation with mouth forming second; positive regulation of ossification; odontogenesis of dentine-containing teeth; formation of radial glial scaffolds; cerebral cortex cell migration; embryonic retina morphogenesis in camera-type eye; neural tube closure; embryonic camera-type eye morphogenesis

Disease: Coronary Artery Disease, Autosomal Dominant 2

NCBI Summary:This gene encodes a member of the low density lipoprotein (LDL) receptor gene family. LDL receptors are transmembrane cell surface proteins involved in receptor-mediated endocytosis of lipoprotein and protein ligands. The protein encoded by this gene functions as a receptor or, with Frizzled, a co-receptor for Wnt and thereby transmits the canonical Wnt/beta-catenin signaling cascade. Through its interaction with the Wnt/beta-catenin signaling cascade this gene plays a role in the regulation of cell differentiation, proliferation, and migration and the development of many cancer types. This protein undergoes gamma-secretase dependent RIP- (regulated intramembrane proteolysis) processing but the precise locations of the cleavage sites have not been determined.[provided by RefSeq, Dec 2009]
UniProt Code:O88572
NCBI GenInfo Identifier:148727288
NCBI Gene ID:4040
NCBI Accession:NP_002327.2
UniProt Secondary Accession:O88572,O88572,
UniProt Related Accession:O75581
Molecular Weight:180,429 Da
NCBI Full Name:low-density lipoprotein receptor-related protein 6
NCBI Synonym Full Names:low density lipoprotein receptor-related protein 6
NCBI Official Symbol:LRP6  
NCBI Official Synonym Symbols:ADCAD2  
NCBI Protein Information:low-density lipoprotein receptor-related protein 6; LRP-6
UniProt Protein Name:Low-density lipoprotein receptor-related protein 6
Protein Family:Low-density lipoprotein receptor-related protein
UniProt Gene Name:LRP6  
UniProt Entry Name:LRP6_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3. Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4. Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6. Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7. Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9. Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10. Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11. Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12. Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13. Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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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