Product Name:	Human Wilms tumor protein (WT1) ELISA Kit
SKU:	HUEB1188
Size:	96T
Target:	Human Wilms tumor protein (WT1)
Synonyms:	WT33
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	15.6-1000pg/mL
Sensitivity:	10pg/ml

Intra CV:

4.6%

Inter CV:

8.3%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	110-120%	92-102%	89-99%	104-116%
EDTA Plasma(N=5)	94-103%	91-101%	104-113%	93-106%
Heparin Plasma(N=5)	93-102%	92-101%	96-105%	94-107%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	89	83-95
Plasma	91	85-97

Function:

Transcription factor that plays an important role in cellular development and cell survival (PubMed:7862533). Recognizes and binds to the DNA sequence 5'-GCG(T/G)GGGCG-3' (PubMed:7862533, PubMed:17716689, PubMed:25258363). Regulates the expression of numerous target genes, including EPO. Plays an essential role for development of the urogenital system. It has a tumor suppressor as well as an oncogenic role in tumor formation. Function may be isoform-specific: isoforms lacking the KTS motif may act as transcription factors (PubMed:15520190). Isoforms containing the KTS motif may bind mRNA and play a role in mRNA metabolism or splicing (PubMed:16934801). Isoform 1 has lower affinity for DNA, and can bind RNA (PubMed:19123921).

Uniprot:	P19544
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Wilms tumor protein
Sub Unit:	Homodimer. Interacts with WTIP. Interacts with actively translating polysomes. Detected in nuclear ribonucleoprotein (mRNP) particles. Interacts with HNRNPU via the zinc-finger region. Interacts with U2AF2. Interacts with CITED2 (By similarity). Interacts with ZNF224 via the zinc-finger region. Interacts with WTAP and SRY. Interacts with AMER1. Interacts with RBM4.
Research Area:	Cancer
Subcellular Location:	Isoform 4 Nucleus Nucleoplasm
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	WT1: a DNA binding protein and apparent transcriptional regulator. Recognizes and binds to the DNA sequence 5'-CGCCCCCGC-3'. Expressed in the kidney and a subset of hematopoietic cells. Defects in WT1 are the cause of Wilms tumor 1 (WT1), an embryonal malignancy of the kidney that affects approximately 1 in 10'000 infants and young children. It occurs both in sporadic and hereditary forms. Four alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:DNA-binding; C2H2-type zinc finger protein; Tumor suppressor; Transcription factor; Nucleolus Chromosomal Location of Human Ortholog: 11p13 Cellular Component: nucleoplasm; cytoplasm; nucleolus; nuclear speck; nucleus Molecular Function:protein binding; zinc ion binding; RNA binding; sequence-specific DNA binding; transcription factor activity Biological Process: tissue development; gonad development; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; heart development; thorax and anterior abdomen determination; negative regulation of transcription from RNA polymerase II promoter; glomerulus development; germ cell development; negative regulation of cell proliferation; regulation of transcription, DNA-dependent; epithelial cell differentiation; ureteric bud development; male genitalia development; vasculogenesis; kidney development; camera-type eye development; transcription, DNA-dependent; adrenal gland development; RNA splicing; glomerular basement membrane development; male gonad development; sex determination; regulation of transcription from RNA polymerase II promoter; ureteric bud branching; negative regulation of translation; positive regulation of transcription from RNA polymerase II promoter; negative regulation of cell growth; negative regulation of transcription, DNA-dependent; negative regulation of apoptosis Disease: Wilms Tumor, Aniridia, Genitourinary Anomalies, And Mental Retardation Syndrome; Denys-drash Syndrome; Mesothelioma, Malignant; Nephrotic Syndrome, Type 4; Frasier Syndrome; Aniridia; Wilms Tumor 1; Meacham Syndrome
NCBI Summary:	This gene encodes a transcription factor that contains four zinc-finger motifs at the C-terminus and a proline/glutamine-rich DNA-binding domain at the N-terminus. It has an essential role in the normal development of the urogenital system, and it is mutated in a small subset of patients with Wilm's tumors. This gene exhibits complex tissue-specific and polymorphic imprinting pattern, with biallelic, and monoallelic expression from the maternal and paternal alleles in different tissues. Multiple transcript variants have been described. In several variants, there is evidence for the use of a non-AUG (CUG) translation initiation site upstream of and in-frame with the first AUG. Authors of PMID:7926762 also provide evidence that WT1 mRNA undergoes RNA editing in human and rat, and that this process is tissue-restricted and developmentally regulated. [provided by RefSeq, Oct 2010]
UniProt Code:	P19544
NCBI GenInfo Identifier:	139778
NCBI Gene ID:	7490
NCBI Accession:	P19544.2
UniProt Secondary Accession:	P19544,Q15881, Q16256, Q16575, Q4VXV4, Q4VXV5, Q4VXV6 Q8IYZ5, A8K6S1, B3KSA5,
UniProt Related Accession:	P19544
Molecular Weight:	449
NCBI Full Name:	Wilms tumor protein
NCBI Synonym Full Names:	Wilms tumor 1
NCBI Official Symbol:	WT1
NCBI Official Synonym Symbols:	GUD; AWT1; WAGR; WT33; NPHS4; WIT-2; EWS-WT1
NCBI Protein Information:	Wilms tumor protein; amino-terminal domain of EWS\|last three zinc fingers of the DNA-binding domain of WT1
UniProt Protein Name:	Wilms tumor protein
UniProt Synonym Protein Names:	WT33
Protein Family:	Wilms tumor protein
UniProt Gene Name:	WT1
UniProt Entry Name:	WT1_HUMAN

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.