Human Transcription factor 7-like 2 (TCF7L2) ELISA Kit (HUEB2467)

Product Name:	Human Transcription factor 7-like 2 (TCF7L2) ELISA Kit
SKU:	HUEB2467
Size:	96T
Target:	Human Transcription factor 7-like 2 (TCF7L2)
Synonyms:	HMG box transcription factor 4, T-cell-specific transcription factor 4, T-cell factor 4, TCF4
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	78-5000pg/mL
Sensitivity:	54pg/mL

Intra CV:

4.8%

Inter CV:

7.5%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	101-111%	96-106%	104-113%	93-103%
EDTA Plasma(N=5)	105-115%	107-116%	99-109%	103-113%
Heparin Plasma(N=5)	101-111%	97-109%	105-114%	96-107%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	104	98-110
Plasma	106	100-112

Function:

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.

Uniprot:	Q9NQB0
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Transcription factor 7-like 2
Sub Unit:	Interacts with TGFB1I1 (By similarity). 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. Interacts with DDIT3/CHOP. The CTNNB1 and TCF7L2/TCF4 complex interacts with PML (isoform PML-4). Identified in a complex with CTNNB1 and FERMT2. Interacts with SPIN1.
Research Area:	Epigenetics
Subcellular Location:	Nucleus PML body Diffuse pattern. Colocalizes with SUMO1 and PIAS4 in a subset of PML (promyelocytic leukemia) nuclear bodies.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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

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.

ELISA

Human TCF7L2 / TCF4 ELISA Kit