Where to buy wnt protein

Upon integration into UniProtKB, each entry is assigned a unique accession number, which is called 'Primary citable accession number'. See complete history. Do not show this banner again. Reactome - a knowledgebase of biological pathways and processes More Reactome i. Recommended name: Protein Wnt-3a. Mus musculus Mouse.

This is known as the 'taxonomic identifier' or 'taxid'. It lists the nodes as they appear top-down in the taxonomic tree, with the more general grouping listed first. MGI i. Does not affect palmitoleoylation. ChEMBL i. N-linked GlcNAc MaxQB i. PaxDb, a database of protein abundance averages across all three domains of life More PaxDb i.

PeptideAtlas More PeptideAtlas i. PRIDE i. ProteomicsDB: a multi-organism proteome resource More ProteomicsDB i. GlyGen i. PhosphoSitePlus i. SwissPalm database of S-palmitoylation events More SwissPalm i. Bgee i. Genevisible search portal to normalized and curated expression data from Genevestigator More Genevisible i.

BioGRID i. CORUM i. Database of interacting proteins More Wnt signaling: a common theme in animal development. The Hedgehog and Wnt signalling pathways in cancer. A new member of the frizzled family from Drosophila functions as a Wingless receptor. Structurally related receptors and antagonists compete for secreted Wnt ligands.

More than one way to skin a catenin. Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, NCCIT. Lab Invest. Wnt-induced dephosphorylation of Axin releases b-catenin from the Axin complex.

Genes Dev. Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless. Mol Cell. LDL-receptor-related protein 6 is a receptor for Dickkopf proteins.

Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells. J Biol Chem. Embo J. Id helix-loop-helix proteins in cell growth and differentiation. Trends Cell Biol. Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins. Dedifferentiation of Mammalian Myotubes Induced by msx1.

REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons. The neuron-restrictive silencer factor NRSF : a coordinate repressor of multiple neuron-specific genes. Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEFdeficient mice. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet. Dissection of an indirect autoregulatory response of a homeotic drosophila gene.

EMBO J. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf Linking colorectal cancer to Wnt signaling. Identification of the receptor component of the IkappaBalpha-ubiquitin ligase.

Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Dfrizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic mutants.

The transcriptional coactivator CBP interacts with beta-catenin to activate gene expression. J Cell Biol. By default, the information is derived from experiments at the mRNA level, unless specified 'at protein level'. Cell Biol. P With Exp. ModBase i Search The information is filed in different subsections. Additionally, this section gives relevant information on each alternative protein isoform. Length: Mass Da : 39, It is useful for tracking sequence updates. The algorithm is described in the ISO standard.

Mass Da : 42, Full view. These are stable identifiers and should be used to cite UniProtKB entries. Upon integration into UniProtKB, each entry is assigned a unique accession number, which is called 'Primary citable accession number'. See complete history. It is not in any way intended to be used as a substitute for professional medical advice, diagnosis, treatment or care.

Do not show this banner again. Pathway Commons web resource for biological pathway data More PathwayCommons i. Reactome - a knowledgebase of biological pathways and processes More Reactome i. SignaLink: a signaling pathway resource with multi-layered regulatory networks More SignaLink i. Recommended name: Protein Wnt-3a.

Homo sapiens Human. This is known as the 'taxonomic identifier' or 'taxid'. It lists the nodes as they appear top-down in the taxonomic tree, with the more general grouping listed first. Human Gene Nomenclature Database More HGNC i. MIM i. VEuPathDB i. DisGeNET i. MalaCards human disease database More MalaCards i. Open Targets More Differentiation towards goblet and enteroendocrine cells was induced by the removal of SB and nicotinamide 3.

Expression of the goblet marker Mucin2 and the enteroendocrine marker chromogranin A was visible within 5 days of induction Fig.

Moreover, in line with previous findings 3 , the Paneth cell marker lysozyme was present both in expansion and differentiation conditions, indicating the presence of Paneth cells in the organoids Fig.

Together, these data show that the organoids derived in Wnt3a liposomes contained multipotent stem cells and were similar to those derived in Wnt3a-conditioned medium with regard to cell types present and similarity to the intestinal crypt. Palmitoylated proteins reversibly interact with lipid bilayers 20 , which may underlie the interaction of Wnt3a with liposomes.

We tested whether the prior association of Wnt protein with liposomes was required for the stabilizing effect in cell culture. However, separate addition of liposomes also prolonged the activity of Wnt3a in a lipid-concentration-dependent manner Fig.

Moreover, separate addition of liposomes and purified Wnt3a supported the clonal expansion of duodenum stem cells Fig. This shows that simple addition of liposomes to organoid medium enhances the stability of Wnt3a protein and allows the serum-free expansion of human organoids.

We explored alternatives for liposomes such as hydrophobic NPs that can be more easily stored and shipped. The NPs stabilized Wnt3a activity to a similar degree as liposomes Fig. On reconstitution, lyophilized Wnt3a liposomes retained most of their activity and physical properties Fig.

Thus, we developed several formats for enhancing Wnt3a stability in serum-free cell culture, depending on the sensitivity of the cell culture system to CHAPS and the final Wnt3a concentration required. Since recombinant Wnt proteins are purified from conditioned media, they contain serum-derived contaminants. While this suggests that some contaminants enhance Wnt protein stability, the high-purity Wnt3a was nonetheless successfully stabilized by liposomes Fig.

The development of serum-free stabilizers to produce Wnt proteins or of alternative molecules that activate the Wnt pathway are potential avenues towards completely eliminating sera.

Recently, it was shown that the serum glycoprotein afamin forms a complex with Wnt3a that remains soluble in aqueous buffer However, we found that recombinant afamin was unable to prolong the activity of purified Wnt3a when present at concentrations found in serum-containing media 23 Supplementary Fig. Possibly, recombinant afamin lacks essential modifications or its stabilization of Wnt3a only occurs when the molecules are complexed during their biosynthesis. However, glycogen synthase kinase 3 inhibitors cannot always substitute for Wnt ligands in the maintenance of stem cells 25 , and we were unable to propagate human duodenum organoids using CHIR in lieu of purified Wnt3a Supplementary Fig.

Since purifying Wnt3a to very high purity lowers yields and thus raises costs, we explored whether the affinity of Wnt3a for liposomes could be exploited to remove contaminants from Wnt3a preparations. These data show that liposomes facilitate not only the stabilization but also the production of high-purity Wnt3a protein. L, molecular weight ladder kDa. Finally, we explored whether Wnt3a liposomes would support the derivation of stem cell cultures from other human organs.

Indeed, Wnt3a liposomes supported organoid derivation from jejunum biopsies Fig. This underscores the inconsistent nature of the Wnt3a-conditioned medium, which was prepared using duodenum-screened serum, and highlights an additional benefit of Wnt3a liposomes as a more universally applicable reagent.

Jejunum organoids were maintained for more than 3 months and displayed robust expansion in the presence of Wnt3a liposomes Supplementary Fig. Furthermore, Wnt3a liposomes supported the derivation of human liver organoids 26 from both three out of four healthy donors and from three cases with end-stage liver disease Fig.

Wnt liposomes hold therefore promise for modelling liver diseases and may have wider applicability. Our study shows that the main technical obstacles with using Wnt protein for serum-free stem cell cultures are its instability and its detergent-associated stem cell toxicity.

The prolonged activity of lipid-stabilized Wnt3a and the absence of CHAPS advance an approach to establish defined long-term cultures of organoids from human organ stem cells.

This removes an obstacle for the use of these cells in clinical scenarios, and lipid-stabilized Wnt3a holds therefore considerable translational potential.

Moreover, the lipid stabilization is compatible with high concentrations of Wnt3a protein and offers advantages for all tissue culture uses of Wnt3a protein. This is demonstrated by the considerably improved performance of Wnt3a liposomes in embryonic stem cell culture. Several approaches have been used to increase the stability and activity of Wnt3a in serum-free conditions.

In one of the first attempts, serum was fractionated and heparan sulfate proteoglycans HSPGs identified as Wnt-stabilizing components Due to their high cost, purified HSPGs are not currently economically viable in cell culture applications. Moreover, the basement membrane extract in which the organ stem cells are cultured is already rich in HSPGs Recently, the serum glycoprotein afamin was identified as another serum component able to stabilize Wnt3a Recombinant afamin did, however, not prolong the activity of purified Wnt3a in our hands.

Since its stabilizing ability was demonstrated in a coexpression setting 22 , this suggests that it depends on the molecules interacting during their biosynthesis or downstream cellular processing. Moreover, this suggests that yet other serum components must be able to stabilize Wnt3a on its dilution in serum-containing medium. This is also indicated by our finding that serum-derived contaminants substantially contribute to Wnt stability, given the inverse correlation that we observed between Wnt3a purity and stability.

Our work adds several other tools to the Wnt instrumentarium: Lipid-coated PLGA NPs can be stored for years 28 , and we show that such particles are as effective as liposomes in prolonging the activity of Wnt3a. Alternatively, for systems that require the elimination of CHAPS, we show that freeze-dried Wnt3a liposomes retain their activity on reconstitution. Finally, liposomes efficiently recover Wnt3a protein from crude preparations while leaving behind most contaminants, which may be applied to improve the economics of Wnt protein purification.

Together, these technologies facilitate distribution of and access to stabilized Wnt ligands. The biopsies were included on availability and none were excluded. The use of donor materials for research purposes was approved by the Medisch Ethische Toetsings Commissies Medical Ethics Committees of Utrecht Medical Center and of Erasmus Medical Center, and informed consent was obtained from all subjects.

After settling of tissue fragments, the supernatant containing crypts was collected, the crypts pelleted, washed with cold chelation buffer and centrifuged at g to remove single cells. Outgrowing crypts of human duodenum and jejunum were typically refreshed every other day. Rspo1- and Noggin-conditioned media were produced by conditioning basal culture medium for 1 week using HEK cells stably transfected with HA-mouse-Rpso1-Fc gift from Calvin Kuo, Stanford University or mouse Noggin-Fc expression vector 29 , respectively.

All cells lines were tested for mycoplasma contamination every 2—3 months. Fractions were analysed for purity and Wnt activity by Coomassie gel and Wnt activity assay see below , and selected fractions were combined for use. Human liver biopsies 0. The use of both donor and recipient materials for research purposes was approved by the Medisch Ethische Toetsings Commissie Medical Ethics Committee of Erasmus Medical Center, and informed consent was obtained from all subjects.

The cell pellet was mixed with Matrigel BD Biosciences and 10, cells were seeded per well in a well plate. The cultures were incubated for 1, 3, 6 and 8 days. The samples were transferred into opaque-walled white well plates for luminescence measurements done on a Centro XS LB Multiplate Luminometer Berthold Technologies. The single cells were cultured in complete culture medium as described above containing the indicated concentration of purified or liposomal Wnt3a or CHIR Tocris; Media were changed every other day.

After 7—10 days, the number of organoids formed was counted. The organoids were again dissociated into single cells and passaged at a dilution that would result in 2, single cells per well again.

The cumulative number of organoids formed at this passage was determined after correcting for the dilution factor used during passaging. The solvent was then evaporated under vacuum on a rotavapor to generate a lipid film. The residual organic solvent was removed by a nitrogen flush. The mean particle size and size distribution polydispersity index of the liposomes were determined with dynamic light scattering using a Malvern Zetasizer.

Purified Wnt3a protein see above was mixed with liposomes at a Phospholipid concentrations were determined by a colorimetric phosphate assay Phospholipids were then degraded by the addition of 0. Evaporation of the perchloric acid was prevented by placing porcelain marbles on top of the tubes. Samples were mixed by vortexing, 0. The pellet was resuspended in PBS and together with the supernatant analysed by western blotting. For each band, background-subtracted quantification numbers, the so-called integrated intensities, were generated with the analysis software provided.

Ratios of integrated intensities were calculated and results were plotted as the percentage of either free or liposome-associated Wnt3a relative to the total amount.