Recombinant Mouse WT1 Protein

• Cat.No.: WT1-18590M
• Product Overview: Recombinant Mouse WT1 full length or partial length protein was expressed.

Specification

• Source: Mammalian Cells
• Species: Mouse
• Tag: His
• Form: Liquid or lyophilized powder
• Endotoxin: < 1.0 EU per μg of the protein as determined by the LAL method.
• Purity: >80%
• Notes: This item requires custom production and lead time is between 5-9 weeks. We can custom produce according to your specifications.
• Storage: Store it at +4 ºC for short term. For long term storage, store it at -20 ºC～-80 ºC.
• Storage Buffer: PBS buffer

Gene Information

• Gene Name: Wt1 Wilms tumor 1 homolog [ Mus musculus ]
• Official Symbol: WT1
• Gene ID: 22431
• mRNA Refseq: NM_144783.2
• Protein Refseq: NP_659032.3
• UniProt ID: P22561

Citation

WT1 Interacts with MAD2 and Regulates Mitotic Checkpoint Function

Journal: Nature communications    PubMed ID: 25232865    Data: 2014/8/5

Authors: Jayasha Shandilya, Eneda Toska, Stefan GE Roberts

Article Snippet:GST, GST-WT1 (and deletion mutants) were expressed in BL21-DE3 competent cells, and His-tagged human MAD2 (and mutant derivatives) were expressed in BL21-DE3(pLysS) competent cells followed by affinity purification as described before .GST, GST-WT1 (and deletion mutants) were expressed in BL21-DE3 competent cells, and His-tagged human MAD2 (and mutant derivatives) were expressed in BL21-DE3(pLysS) competent cells followed by affinity purification as described before .. Flag-tagged full length WT1 protein (residue 1–449, including exon 5/17AA and KTS) was purchased from Creative BioMart.. The MAD2-binding peptide 1 (MBP1) was purchased from Peptide 2.0 (sequence: SWYSYPPPQRAV); Control peptide sequence: CKATKDPSRVGDS.The MAD2-binding peptide 1 (MBP1) was purchased from Peptide 2.0 (sequence: SWYSYPPPQRAV); Control peptide sequence: CKATKDPSRVGDS.

(a) In vitro interaction assay was performed with either GST-WT1 (residues 245–297) or GST in the presence of full length His-MAD2. The interaction was analyzed by immunoblotting with anti-MAD2 antibody. Bound proteins were also resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. (b) In vitro pulldown assay was also performed with either Flag-M2 magnetic beads alone or incubated with full length Flag-tagged WT1 protein in presence of His-MAD2. The interaction was analyzed by immunoblotting with anti-WT1 and anti-MAD2 antibodies (c) GST-interaction assay was carried out with WT1 containing the 17 amino acid insertion (residues 180-297, +17AA) or lacking it (residues 180-297, Δ17 AA) with His-MAD2. The interaction was analyzed by immunoblotting with anti-MAD2 antibody. Bound proteins were also resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. (d) HeLa cells were transfected with GFP Vector, full length GFP-WT1 (?/?) or GFP-WT1 (?/+) isoforms and 48 hours later whole cell extracts were prepared followed by immunoprecipitation with anti-MAD2 antibodies. The immunoprecipitates were probed with anti-WT1 antibody. Blotting with anti-MAD2 antibody was performed as a control. (e) The MAD2 interaction with ectopically expressed GFP tagged full length WT1 (+/+) and WT1 (+/?) isoforms was analyzed as in part d (f) HeLa cells were transfected with pcDNA vector, pcDNA driving expression of WT1?/?DDS (R394X) or wild type WT1?/? followed by immunoprecipitation with anti-MAD2 antibodies. The immunoprecipitates were probed with anti-WT1 antibodies. Blotting with anti-MAD2 antibodies was performed as a control.

(a) GST-interaction assays were performed with the internal deletion mutants of GST-WT1 (schematic) with full length His-MAD2 and immunoblotted with anti-MAD2 and anti-GST antibodies. (b) Co-immunoprecipitation assays were carried out with whole cell extracts derived from HeLa cells that had been transfected with either GFP vector, GFP WT1 (?/?) or GFP-WT1 (?/?Δ4) using anti-MAD2 or anti-WT1 antibodies. (c) Co-immunofluorescence analysis was carried out in WiT49 cells with anti-MAD2 and anti-WT1 antibodies at the pro-metaphase stage of mitosis. DNA was stained with Hoechst. The scale bar is 10 microns. (d) M15, WiT49 and K562 cells were treated with 60 ng/ml nocodazole for 24 hours, whole cell extracts prepared and immunoprecipitation performed with anti-MAD2 antibodies followed by immunoblotting with WT1 antibody. Input is 10% of the total whole cell extract.

(a) GST-interaction assays were carried out with WT1 (residues 245–297) and full length (FL) His-MAD2 alone and also with MAD2 pre-incubated with either 40μM of MAD2-binding peptide 1 (MBP1), or a control peptide. The pulldown complexes were resolved by SDS-PAGE and immunoblotted with anti-MAD2 antibodies. (b) In vitro pulldown assays were performed with either Flag-M2 magnetic beads alone or full length Flag-tagged WT1 protein in the presence of Full length His-MAD2 (pre-incubated with MBP1) or the MAD2 delC mutant. The interaction was analyzed by immunoblotting with anti-WT1 and anti-MAD2 antibodies. (c) GST-interaction assays were carried out with His-MAD2, MAD2 L13A and MAD2-C-terminal deletion mutant (Del-C). Immunoblotting was performed using anti-MAD2 and anti- GST antibodies. (d) GST-interaction assays were carried out with His-MAD2 + MBP1, MAD2-Del-C, and MAD2 dimerization mutant, R133A. Immunoblotting was performed using anti-MAD2 and anti-GST antibodies. (e) Pull down assays were performed with full length Flag-WT1 protein and the different MAD2 point mutant derivatives. Immunoblotting was performed using anti-MAD2 and anti-WT1 antibodies. Input is 20% of the different MAD2 proteins used for interaction analysis.

Reviews

10/10/2020

It has been widely employed in protein electron microscopy structure analysis, where it has demonstrated remarkable efficacy.

10/25/2019

Broad applicability make it a valuable asset for a wide range of research endeavors.

05/07/2017

Researchers can rely on the WT1 Protein to deliver reliable and reproducible results, facilitating robust data interpretation and scientific advancements.

Q&As

What therapeutic strategies are being explored for cancers with abnormal WT1 expression?

Targeted therapies and immunotherapies aimed at inhibiting or modulating WT1 expression are being investigated for their potential in cancer treatment.

In what other cancers is the WT1 protein targeted for diagnostic purposes?

Apart from Wilms' tumor, WT1 is examined in leukemia, mesothelioma, and some solid tumors as a diagnostic marker.

Can WT1 be a target for personalized cancer therapy?

Yes, understanding the WT1 status in a patient's cancer can guide the development of personalized treatment plans, potentially improving outcomes.

Are there any ongoing clinical trials focused on the WT1 protein and cancer?

Yes, there are several clinical trials exploring the role of WT1 as a therapeutic target and its potential in cancer treatment.

How does the WT1 protein contribute to the prognosis of cancer patients?

High levels of WT1 expression are often associated with poor prognosis in various cancers, indicating a more aggressive disease course.