Product Name:	Human WT1 / Wilms Tumor Protein ELISA Kit
Product Code:	HUFI01056
Size:	96 Assays
Alias:	WT1, Wilms tumor protein, GUD, WAGR, WIT-2, WT33, AWT1, NPHS4GUD, WAGR, Wilms tumor 1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human WT1 concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human WT1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human WT1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-93	90
EDTA plasma(n=5)	92-104	97
UFH plasma(n=5)	89-104	98

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human WT1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	92-104%	85-103%	91-103%
EDTA plasma(n=5)	86-97%	85-96%	85-95%
UFH plasma(n=5)	82-99%	85-98%	80-98%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

P19544

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 EWSlast 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

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.