Human TCF7L2/TCF4 ELISA Kit (HUFI01972)

Product Name:	Human TCF7L2/TCF4 ELISA Kit
Product Code:	HUFI01972
Size:	96 Assays
Alias:	TCF7L2, HMG box transcription factor 4, T-cell-specific transcription factor 4, T-cell factor 4, TCF-4, hTCF-4, TCF7L2, TCF4
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human TCF7L2 concentrations in serum plasma and other biological fluids.
Sensitivity:	46.875pg/ml
Range:	78.125-5000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human TCF7L2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human TCF7L2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-103	95
EDTA plasma(n=5)	85-102	91
UFH plasma(n=5)	87-101	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TCF7L2 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)	94-104%	85-105%	86-96%
EDTA plasma(n=5)	83-91%	86-101%	82-101%
UFH plasma(n=5)	80-99%	84-100%	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

Q9NQB0

UniProt Protein Function:	TCF7L2: Participates in the Wnt signaling pathway and modulates MYC expression by binding to its promoter in a sequence-specific manner. Acts as repressor in the absence of CTNNB1, and as activator in its presence. Activates transcription from promoters with several copies of the Tcf motif 5'-CCTTTGATC-3' in the presence of CTNNB1. TLE1, TLE2, TLE3 and TLE4 repress transactivation mediated by TCF7L2/TCF4 and CTNNB1. Expression of dominant-negative mutants results in cell-cycle arrest in G1. Necessary for the maintenance of the epithelial stem-cell compartment of the small intestine. Interacts with TGFB1I1. Interacts with CTNNB1 (via the armadillo repeat); forms stable transcription complex. Interacts with EP300. Interacts with NLK. Interacts with CCDC85B (probably through the HMG box); prevents interaction with CTNNB1. Interacts with TNIK. Interacts with MAD2L2; prevents TCF7L2/TCF4 binding to promZIPK/DAPK3oters, negatively modulating its transcriptional activity. Interacts with ZIPK/DAPK3. Interacts with XIAP/BIRC4 and TLE3. Detected in epithelium from small intestine, with the highest expression at the top of the crypts and a gradient of expression from crypt to villus. Detected in colon epithelium and colon cancer, and in epithelium from mammary gland and carcinomas derived therefrom. Belongs to the TCF/LEF family. 10 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Apoptosis; Motility/polarity/chemotaxis; DNA-binding; Transcription factor; Cell development/differentiation; Cell cycle regulation Chromosomal Location of Human Ortholog: 10q25.3 Cellular Component: nucleoplasm; transcription factor complex; PML body; cytoplasm; nuclear chromatin; nucleus Molecular Function:protein binding; sequence-specific DNA binding; gamma-catenin binding; beta-catenin binding; chromatin binding; transcription factor binding; transcription factor activity; protein kinase binding; nuclear hormone receptor binding Biological Process: neural tube development; skin development; glycogen metabolic process; fat cell differentiation; regulation of myelination; somatic stem cell maintenance; positive regulation of apoptosis; Wnt receptor signaling pathway through beta-catenin; glucose homeostasis; maintenance of DNA repeat elements; negative regulation of BMP signaling pathway; post-embryonic development; embryonic digestive tract morphogenesis; response to glucose stimulus; cell cycle arrest; oligodendrocyte development; embryonic hindgut morphogenesis; embryonic genitalia morphogenesis; regulation of hormone metabolic process; transcription, DNA-dependent; regulation of smooth muscle cell proliferation; negative regulation of transcription factor activity; glucose metabolic process; positive regulation of insulin secretion; cellular response to starvation; negative regulation of organ growth; negative regulation of fat cell differentiation; regulation of transcription from RNA polymerase II promoter; positive regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; positive regulation of epithelial cell proliferation; secretory granule localization; myoblast cell fate commitment; positive regulation of protein binding; negative regulation of fibroblast growth factor receptor signaling pathway; regulation of oligodendrocyte differentiation; negative regulation of transcription from RNA polymerase II promoter; bone mineralization; positive regulation of protein export from nucleus; positive regulation of gluconeogenesis; pancreas development; blood vessel development; multicellular organism growth; odontogenesis of dentine-containing teeth; positive regulation of protein kinase B signaling cascade; cell proliferation; generation of neurons; pituitary gland development; regulation of skeletal muscle development; brain development Disease: Diabetes Mellitus, Noninsulin-dependent
NCBI Summary:	This gene encodes a high mobility group (HMG) box-containing transcription factor that plays a key role in the Wnt signaling pathway. The protein has been implicated in blood glucose homeostasis. Genetic variants of this gene are associated with increased risk of type 2 diabetes. Several transcript variants encoding multiple different isoforms have been found for this gene.[provided by RefSeq, Oct 2010]
UniProt Code:	Q9NQB0
NCBI GenInfo Identifier:	29337146
NCBI Gene ID:	6934
NCBI Accession:	Q9NQB0.2
UniProt Secondary Accession:	Q9NQB0,B4DRJ8, B9X074, C6ZRJ8, C6ZRK0, E2GH14, E2GH19 E2GH20, E2GH24, E2GH25, E9PFH9, F8W742,
UniProt Related Accession:	Q9NQB0
Molecular Weight:	619
NCBI Full Name:	Transcription factor 7-like 2
NCBI Synonym Full Names:	transcription factor 7-like 2 (T-cell specific, HMG-box)
NCBI Official Symbol:	TCF7L2
NCBI Official Synonym Symbols:	TCF4; TCF-4
NCBI Protein Information:	transcription factor 7-like 2; hTCF-4; T-cell factor 4; T-cell factor-4 variant A; T-cell factor-4 variant B; T-cell factor-4 variant C; T-cell factor-4 variant D; T-cell factor-4 variant E; T-cell factor-4 variant F; T-cell factor-4 variant G; T-cell fac
UniProt Protein Name:	Transcription factor 7-like 2
UniProt Synonym Protein Names:	HMG box transcription factor 4; T-cell-specific transcription factor 4; T-cell factor 4; TCF-4; hTCF-4
Protein Family:	Transcription factor
UniProt Gene Name:	TCF7L2
UniProt Entry Name:	TF7L2_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 Âµl 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 Âµl 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.

Human TCF7L2/TCF4 ELISA Kit (HUFI01972)

Description