Mouse Thyroid transcription factor 1/TITF1 ELISA Kit (MOFI01154)

Product Name:	Mouse Thyroid transcription factor 1/TITF1 ELISA Kit
Product Code:	MOFI01154
Size:	96 Assays
Alias:	TITF1
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse TITF1 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse TITF1 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse TITF1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-102	94
EDTA plasma(n=5)	87-98	92
UFH plasma(n=5)	85-101	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse TITF1 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)	86-105%	85-104%	89-96%
EDTA plasma(n=5)	82-97%	86-100%	85-100%
UFH plasma(n=5)	80-91%	81-99%	80-95%

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

P50220

UniProt Protein Function:	NKX2-1: Transcription factor that binds and activates the promoter of thyroid specific genes such as thyroglobulin, thyroperoxidase, and thyrotropin receptor. Crucial in the maintenance of the thyroid differentiation phenotype. May play a role in lung development and surfactant homeostasis. Defects in NKX2-1 are the cause of benign hereditary chorea (BHC); also known as hereditary chorea without dementia. BHC is an autosomal dominant movement disorder. The early onset of symptoms (usully before the age of 5) and the observation that in some BHC families the symptoms tend to decrease in adulthood suggests that the disorder results from a developmental disturbance of the brain. BHC is non-progressive and patients have normal or slightly below normal intelligence. There is considerable inter- and intrafamilial variability, including dysarthria, axial distonia and gait disturbances. Defects in NKX2-1 are the cause of choreoathetosis, hypothyroidism, and neonatal respiratory distress (CHNRD). This syndrome include neurological, thyroid, and respiratory problems. Belongs to the NK-2 homeobox family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 14q13 Cellular Component: nucleoplasm; transcription factor complex; nucleus Molecular Function:RNA polymerase II transcription factor activity, enhancer binding; protein binding; enzyme binding; DNA binding; sequence-specific DNA binding; transcription factor activity Biological Process: transcription from RNA polymerase II promoter; axon guidance; positive regulation of transcription, DNA-dependent; rhythmic process; neuron migration; response to hormone stimulus; globus pallidus development; locomotory behavior; forebrain dorsal/ventral pattern formation; negative regulation of transcription from RNA polymerase II promoter; positive regulation of circadian rhythm; Leydig cell differentiation; phospholipid metabolic process; forebrain development; thyroid gland development; oligodendrocyte differentiation; anatomical structure formation; forebrain neuron fate commitment; negative regulation of cell migration; hippocampus development; induction; cerebral cortex GABAergic interneuron differentiation; cerebral cortex cell migration; pituitary gland development; positive regulation of transcription from RNA polymerase II promoter; brain development; negative regulation of transcription, DNA-dependent; negative regulation of transforming growth factor beta receptor signaling pathway; endoderm development; lung development Disease: Thyroid Carcinoma, Papillary; Chorea, Benign Hereditary; Choreoathetosis And Congenital Hypothyroidism With Or Without Pulmonary Dysfunction
NCBI Summary:	This gene encodes a protein initially identified as a thyroid-specific transcription factor. The encoded protein binds to the thyroglobulin promoter and regulates the expression of thyroid-specific genes but has also been shown to regulate the expression of genes involved in morphogenesis. Mutations and deletions in this gene are associated with benign hereditary chorea, choreoathetosis, congenital hypothyroidism, and neonatal respiratory distress, and may be associated with thyroid cancer. Multiple transcript variants encoding different isoforms have been found for this gene. This gene shares the symbol/alias 'TTF1' with another gene, transcription termination factor 1, which plays a role in ribosomal gene transcription. [provided by RefSeq, Feb 2014]
UniProt Code:	P50220
NCBI GenInfo Identifier:	1174819
NCBI Gene ID:	7080
NCBI Accession:	P43699.1
UniProt Secondary Accession:	P50220,P50220, P23441,
UniProt Related Accession:	P43699
Molecular Weight:	401
NCBI Full Name:	Homeobox protein Nkx-2.1
NCBI Synonym Full Names:	NK2 homeobox 1
NCBI Official Symbol:	NKX2-1
NCBI Official Synonym Symbols:	BCH; BHC; NK-2; TEBP; TTF1; NKX2A; T/EBP; TITF1; TTF-1; NKX2.1
NCBI Protein Information:	homeobox protein Nkx-2.1; NK-2 homolog A; thyroid nuclear factor 1; thyroid transcription factor 1; homeobox protein NK-2 homolog A; thyroid-specific enhancer-binding protein
UniProt Protein Name:	Homeobox protein Nkx-2.1
UniProt Synonym Protein Names:	Homeobox protein NK-2 homolog A; Thyroid nuclear factor 1; Thyroid transcription factor 1; TTF-1; Thyroid-specific enhancer-binding protein; T/EBP
UniProt Gene Name:	NKX2-1
UniProt Entry Name:	NKX21_HUMAN

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

Step	Procedure
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.

Mouse Thyroid transcription factor 1/TITF1 ELISA Kit (MOFI01154)

Description