Overview

  • Product name

    Anti-LRP6 antibody [1C10]
    See all LRP6 primary antibodies
  • Description

    Mouse monoclonal [1C10] to LRP6
  • Host species

    Mouse
  • Specificity

    This antibody shows no cross reactivity with LRP5.
  • Tested applications

    Suitable for: WB, IPmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Cytoplasmic domain of human LRP6.

Properties

Applications

Our Abpromise guarantee covers the use of ab75358 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000 - 1/2000. Detects a band of approximately 180 kDa (predicted molecular weight: 179 kDa).
IP 1/1000 - 1/2000.

Target

  • Function

    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.
  • Tissue specificity

    Widely co-expressed with LRP5 during embryogenesis and in adult tissues.
  • Involvement in disease

    Defects in LRP6 are the cause of autosomal dominant coronary artery disease type 2 (ADCAD2) [MIM:610947].
  • Sequence similarities

    Belongs to the LDLR family.
    Contains 4 EGF-like domains.
    Contains 3 LDL-receptor class A domains.
    Contains 20 LDL-receptor class B repeats.
  • Domain

    The YWTD-EGF-like domains 1 and 2 are required for the interaction with Wnt-frizzled complex. The YWTD-EGF-like domains 3 and 4 are required for the interaction with DKK1.
    The PPPSP motifs play a central role in signal transduction by being phosphorylated, leading to activate the Wnt signaling pathway.
  • Post-translational
    modifications

    Dual phosphorylation of cytoplasmic PPPSP motifs sequentially by GSK3 and CK1 is required for AXIN1-binding, and subsequent stabilization and activation of beta-catenin via preventing GSK3-mediated phosphorylation of beta-catenin. Phosphorylated, in vitro, by GRK5/6 within and outside the PPPSP motifs. Phosphorylation at Ser-1490 by CDK14 during G2/M phase leads to regulation of the Wnt signaling pathway during the cell cycle. Phosphorylation by GSK3B is induced by RPSO1 binding and inhibited by DKK1. Phosphorylated, in vitro, by casein kinase I on Thr-1479.
    Undergoes gamma-secretase-dependent regulated intramembrane proteolysis (RIP). The extracellular domain is first released by shedding, and then, through the action of gamma-secretase, the intracellular domain (ICD) is released into the cytoplasm where it is free to bind to GSK3B and to activate canonical Wnt signaling.
    Palmitoylation on the two sites near the transmembrane domain leads to release of LRP6 from the endoplasmic reticulum.
    Mono-ubiquitinated which retains LRP6 in the endoplasmic reticulum.
    N-glycosylation is required for cell surface location.
  • Cellular localization

    Membrane. Endoplasmic reticulum. On Wnt signaling, undergoes a cycle of caveolin- or clathrin-mediated endocytosis and plasma membrane location. Released from the endoplasmic reticulum on palmitoylation. Mono-ubiquitination retains it in the endoplasmic reticulum in the absence of palmitoylation. On Wnt signaling, phosphorylated, aggregates and colocalizes with AXIN1 and GSK3B at the plasma membrane in LRP6-signalsomes. Chaperoned to the plasma membrane by MESD.
  • Information by UniProt
  • Database links

  • Alternative names

    • ADCAD2 antibody
    • C030016K15Rik antibody
    • Cd antibody
    • FLJ90062 antibody
    • FLJ90421 antibody
    • LDL receptor related protein 6 antibody
    • Low density lipoprotein receptor related protein 6 antibody
    • Low-density lipoprotein receptor-related protein 6 antibody
    • LRP-6 antibody
    • LRP6 antibody
    • LRP6_HUMAN antibody
    • OTTHUMP00000238979 antibody
    • OTTHUMP00000238980 antibody
    • OTTHUMP00000238982 antibody
    • STHAG7 antibody
    see all

Images

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: LRP6 knockout HAP1 cell lysate (20 µg)
    Lane 3: HeLa cell lysate (20 µg)
    Lane 4: HepG2 cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab75358 observed at 200, 220 kDa. Red - loading control, ab18251, observed at 52 kDa.

    ab75358 was shown to specifically react with LRP6 when LRP6 knockout samples were used. Wild-type and LRP6 knockout samples were subjected to SDS-PAGE. ab75358 and ab18251 (loading control to alpha Tubulin) were diluted at 1/1000  and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

  • Western blot (100ug) and immunoprecipitation (2mg) analysis of total cell lysates from 293T cells using ab75358.

References

This product has been referenced in:

  • Agajanian MJ  et al. WNT Activates the AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback Loop. Cell Rep 26:79-93.e8 (2019). Read more (PubMed: 30605688) »
  • Yang Q  et al. Knockdown of SSATX, an alternative splicing variant of the SAT1 gene, promotes melanoma progression. Gene 716:144010 (2019). Read more (PubMed: 31352009) »
See all 11 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question

DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE Mouse embryonic fibroblasts and HC11 cell line/membrane/cytosolic extract/ endogenous protein PRIMARY ANTIBODY Incubate overnight with Lrp6 at 1:1000, 1:500, 1:250 dilutions in 5% milk TBS-T at 4 degrees C on a rocker. Wash 3 times in TBS-T buffer for 5 min. per wash on shaker. DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Positive control: recombinant Lrp6 protein from transfected 293 cells. Negative control: KO cells ANTIBODY STORAGE CONDITIONS Aliquoted and stored at -80 C. Aliquots upon thawing, stored at 4 C. SAMPLE PREPARATION Non-denaturing lysis buffer with 1 % Triton X-100 and 1x protease inhibitor cocktail and 1x phosphatase inhibitor cocktail. Cells were scraped in lysis buffer and cell debris was spun down at 14,000 rpm for 15 min at 4 C. Supernatants were transferred to new tube and bradford assay was performed. For protein loading, protein solution was boiled for 5 min in 1x Sample loading buffer AMOUNT OF PROTEIN LOADED 25 ug per lane. ELECTROPHORESIS/GEL CONDITIONS Reducing gel, 4-20%, ran at 40 mA for 1:30 hours TRANSFER AND BLOCKING CONDITIONS Transfer buffer: 48 mM Tris base, 39 mM glycine. Transfer to PVDF membrane with 100 V for one hour. Blocking agent: 5% milk in TBS-tween buffer. SECONDARY ANTIBODY goat anti-mouse/5 % milk TBS-T/ 1:5000, 1:2500 dilution/ 1-2 hours at room temperature. 3 x TBS-T wash for 5 min per wash on shaker WHAT STEPS HAVE YOU ALTERED? increasing the primary and secondary antibody amounts as well as increasing the amount of protein loaded on the gel. Also, harvesting fresh lysates from different batches of wild-type primary MEFs ADDITIONAL NOTES Positive control works but we used to be able to detect endogenous Lrp6 protein in MEFs at a 1:1000 dilution of primary antibody using the exact conditions described above (lysis, gel, and transfer conditions) The last 2 vials of this lot we have purchased has given us very weak signal if any signal for endogenous protein detection. Have other labs seen weak signal with this lot of antibody? Thank you for any advice you can give me!

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Answer

Thank you for your inquiry. I am sorry that you have been experiencing difficulties with this antibody in WB. I have reviewed your protocol information and also checked for other problems reported with this antibody. We have not received many reports of this product showing no signal in WB (only one other but was resolved by protocol tips) and this is a good selling antibody. Since you do get a signal in your positive control, I would advise increasing the amount of lysate that you load. We typically recommend 60 - 100ug of total protein. Additionally, consider decreasing the percentage of milk used or using BSA instead to block and use as antibody diluent. Have you confirmed efficient transfer of your proteins? Such large proteins are more difficult to transfer and if only small amounts of protein are transferred, it may be necessary to modify the conditions to improve the efficiency. I would recommend adding SDS to 0.1% and reducing the methanol to 10% or less in your transfer buffer to improve your results. I hope that you find these suggestions helpful. Please do not hesitate to contact us if you have any additional questions or if these suggestions do not improve your results with this antibody.

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Answer

Thank you for your inquiry. I can confirm that ab75358 (Anti-LRP6 antibody [1C10]) will bind to the intracellular region of LRP6. The immunogen for this antibody was the cytoplasmic domain of human LRP6. Unfortunately, this monoclonal antibody has not been mapped to a narrower range of amino acids. I hope this information is helpful. Please do not hesitate to contact me again with any further questions.

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Answer

Thank you for your phone call yesterday and for your patience while I have looked into your question. I have heard back from the lab, and I was told that the LRP6 mab [1C10] has been tested against denatured LRP6 by western blotting and non-denatured LRP6 by IP. I hope this information is helpful, but please let me know if you have any further questions.

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