WT1 Antibody (6F-H2)

Western Blot: WT1 Antibody (6F-H2) [NB110-60011] - Hypomethylation of the Intron 1 CpG island results in WT1 expression. RNA was isolated from K562 and U937 cells growing under atmospheric conditions (Normox) and from U937 cells growing in 1% O2 (Hypox) and was analyzed by RT-PCR using primers that span exon 5 of the WT1 mRNA. Ribosomal RNA 36B4 is used as a positive control, and molecular weight markers (MW) are shown. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0119837), licensed under a CC-BY licence.

Immunohistochemistry-Paraffin: WT1 Antibody (6F-H2) [NB110-60011] - Staining of human kidney glomerulus tissue. Heat mediated antigen retrieval was performed by heating in citrate buffer (pH 6) at 95C for 20 minutes. Image from verified customer review.

Western Blot: WT1 Antibody (6F-H2) [NB110-60011] - Whole cell protein from human K562, HEK293 and kidney tissue was separated on a 12% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 2.0 ug/ml anti-WT1 in block buffer and detected with an anti-mouse HRP secondary antibody using chemiluminescence.

Immunohistochemistry: WT1 Antibody (6F-H2) [NB110-60011] - Analysis of Wilms Tumor 1 in human renal cancer using DAB with hematoxylin counterstain.

Simple Western: WT1 Antibody (6F-H2) [NB110-60011] - Lane view shows a specific band for WT1 in 0.5 mg/ml of Hek293 lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

• Reactivity: Hu
• Applications: WB, Simple Western, ICC/IF, IHC, IHC-Fr, IHC-P, IP
• Clone: 6F-H2
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Concentration: 1.0 mg/ml

WT1 Antibody (6F-H2) Summary

• Immunogen: Human recombinant Wilms Tumor 1 protein (WT1) (residues 1-181). [Swiss-Prot: P19544]
• Localization: Nuclear, Cytoplasm
• Isotype: IgG1 Kappa
• Clonality: Monoclonal
• Host: Mouse
• Gene: WT1
• Purity: Protein G purified

Applications/Dilutions

• Dilutions:
  • Western Blot 2 ug/ml
  • Simple Western 1:50
  • Immunocytochemistry/Immunofluorescence
  • Immunohistochemistry 1:400
  • Immunohistochemistry-Frozen 1: 400
  • Immunohistochemistry-Paraffin 1:400
  • Immunoprecipitation 2-10 ug/ml lysate
• Application Notes: In Western blot, a doublet is observed at approx. 55 kDa. Prior to immunostaining paraffin tissues, antigen retrieval with sodium citrate buffer (pH 6.0) is recommended. In immunostaining, this target may be found localized to nucleus/nucleolus and cytoplasm of cells (WT1 shuttles between nucleus and cytoplasm - PMID: 14681305). In Simple Western only 10 - 15 uL of the recommended dilution is used per data point. Separated by Size-Wes, Sally Sue/Peggy Sue. Use in Immunocytochemistry/immunofluorescence reported in scientific literature (PMID: 25576053).
• Theoretical MW: 55 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• Control:

  293 Whole Cell Lysate
  K-562 Whole Cell Lysate
  WT1 Overexpression Lysate

Packaging, Storage & Formulations

• Storage: Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.
• Buffer: PBS
• Preservative: 0.02% Sodium Azide
• Concentration: 1.0 mg/ml
• Purity: Protein G purified

Alternate Names for WT1 Antibody (6F-H2)

• AWT1
• GUD
• NPHS4GUD
• WAGR
• Wilms tumor 1
• Wilms tumor protein
• WIT-2
• WT1
• WT33

Background

Wilms' tumor 1 (WT1) gene inactivation is responsible for almost 20% of Wilms' tumour/developmental renal tumour or nephroblastoma cases and besides playing important role in cellular development and survival, this homodimeric transcription factor has been shown to involve in certain pathologies also. WT1 belongs to EGR C2H2-type zinc-finger protein family and interacts with several proteins including WTIP, HNRNPU, U2AF2, CITED2, ZNF224, WTAP, SRY, AMER1, RBM4 and actively translating polysomes (detected in nuclear ribonucleoprotein particles) for exerting its activities. WT1 binds to DNA sequence 5'-CGCCCCCGC-3' and regulates the expression of several target genes including EPO. It plays essential role in urogenital system development and in carcinogenesis, it has been suggested as tumor suppressor as well as oncogenic protein. Defects in WT1 have been linked to Wilms tumor 1 (WT1), Frasier syndrome (FS), Denys-Drash syndrome (DDS), nephrotic syndrome type 4 (NPHS4), Meacham syndrome (MEACHS), mesothelioma malignant (MESOM).

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for WT1 Antibody (NB110-60011)(4)

Publications using NB110-60011

• McCarty G, Loeb DM. Hypoxia-Sensitive Epigenetic Regulation of an Antisense-Oriented lncRNA Controls WT1 Expression in Myeloid Leukemia Cells. PLoS ONE. 2015 Mar 21 [PMID: 25794157] (WB, Human)
  
  Details:
  WT1 antibody used for WB on total cell lysates from K562, U937 and HL60 that were treated with CoCl2, 5-azacytidine, or untreated controls (Figure 2C).WT1 antibody used at a dilution of 1:1,000 for WB on total cell lysates from K562, U937 and HL60 that were treated with CoCl2, 5-azacytidine, or untreated controls (Figure 2C).
• Jeitany M, Pineda JR, Liu Q et al. A preclinical mouse model of glioma with an alternative mechanism of telomere maintenance (ALT). Int. J Cancer. 2014 Aug 30 [PMID: 25175359]
• McCarty G, Awad O, Loeb DM. WT1 directly regulates expression of vascular endothelial growth factor and is a mediator of tumor response to hypoxia. The Journal of Biological Chemistry. 2011 Oct 26. [PMID: 22030397]
• Rauscher III, FJ et al. Characterization of monoclonal antibodies directed to the amino-terminus of the WT1, Wilms' tumor suppressor protein. Hybridoma;17(2):191-8. 1998 Apr. [PMID: 9627060]

Reviews for WT1 Antibody (NB110-60011) (3)

Average Rating: 4.3

(Based on 3 reviews)

We have 3 reviews tested in 2 species: Human, Mouse and Human.

reviewed by: Anonymous WB Mouse and Human 10/17/2019

Summary

• Application: Western Blot
• Sample Tested: Liver,293T whole cell lysate
• Species: Mouse and Human
• Lot: B-4

reviewed by: Santhosh Sivajothi IHC-P Human 05/22/2018

Summary

• Application: Immunohistochemistry-Paraffin
• Sample Tested: Human kidney tissue
• Species: Human
• Lot: A-5

Comments

• Comments: Heat mediated antigen retrieval was performed by heating in citrate buffer (pH6) at 95C for 20 minutes

reviewed by: Evren Azeloglu WB Human 04/28/2014

Summary

• Application: Western Blot
• Sample Tested: Immortalized podocytes
• Species: Human

Blocking

• Blocking Details: Rockland casein blocking buffer, 2 hrs, RT

Primary Anitbody

• Dilution Ratio: 1:1000 in 5% milk in TBS-T, overnight, 4ºC

Secondary Antibody

• Secondary Description: Thermo goat anti-rabbit HRP
• Secondary Concentration: 1:2000 in 5% milk in TBS-T, 30min, RT

Details

• Detection Notes: Thermo ECL, 2min

FAQs for WT1 Antibody (NB110-60011). (Showing 1 - 2 of 2 FAQs).

1. I just wondering this antibody (WT1 antibody (6F-H2)[Alexa Flour488], when I use it to staining tissue do I need incubate in second antibody or not?
   • Our product NB110-60011AF488 is directly conjugated with an Alexa Fluor 488 dye, so you do not need to use any secondary antibody with this product to detect the antibody. The dye labeled to this antibody excites at 490nm and emits at 525nm.
2. In NB800-PC6, were the 293 cells transfected with WT1 expression plasmids? We have 293 cell lysates and it showed negative WT1 expression. Just want to make sure if our antibody is not working.
   • This protein lysate was derived from human HEK293 cells (without any transfection) and it is a whole cell lysate. WT1's mRNA is expressed significantly in HEK294 cells protein atlas site and given the fact that our Wilms Tumor 1 antibody (6F-H2) [NB110-60011] showed positive bands on HEK293 cells lysate, you should be good for considering this lysate as a positive control for WT1.

Bioinformatics

• Gene Symbol: WT1