ねずみ HNF1a ELISA (MOFI00895)

商品名:	Mouse HNF1a / Hepatocyte Nuclear Factor 1 Alpha ELISA Kit
製品コード:	MOFI00895
サイズ:	96 Assays
エイリアス:	HNF1a
検出方法:	Sandwich ELISA
申し込み:	This immunoassay kit allows for the in vitro quantitative determination of Mouse HNF1a concentrations in serum plasma and other biological fluids.
感度:	4.688pg/ml
範囲:	7.813-500pg/ml
保管所:	4°C for 6 months
ノート:	For Research Use Only

回復:

Matrices listed below were spiked with certain level of Mouse HNF1a and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse HNF1a in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-101	90
EDTA plasma(n=5)	88-100	93
UFH plasma(n=5)	90-103	96

直線性:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse HNF1a 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)	90-105%	91-104%	91-103%
EDTA plasma(n=5)	82-97%	89-94%	83-98%
UFH plasma(n=5)	85-99%	83-98%	86-96%

Intra Assay:

CV <8%

Inter Assay:

CV <10%

成分	量	保管所
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	-

必要なその他の材料と設備：

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

P22361

UniProt Protein Function:	HNF1A: Transcriptional activator that regulates the tissue specific expression of multiple genes, especially in pancreatic islet cells and in liver. Required for the expression of several liver specific genes. Binds to the inverted palindrome 5'- GTTAATNATTAAC-3'. Defects in HNF1A are a cause of hepatic adenomas familial (HEPAF). Hepatic adenomas are rare benign liver tumors of presumable epithelial origin that develop in an otherwise normal liver. Hepatic adenomas may be single or multiple. They consist of sheets of well-differentiated hepatocytes that contain fat and glycogen and can produce bile. Bile ducts or portal areas are absent. Kupffer cells, if present, are reduced in number and are non-functional. Conditions associated with adenomas are insulin-dependent diabetes mellitus and glycogen storage diseases (types 1 and 3). Bi-allelic inactivation of HNF1A, whether sporadic or associated with MODY3, may be an early step in the developmant of some hepatocellular carcinomas. Defects in HNF1A are the cause of maturity-onset diabetes of the young type 3 (MODY3); also symbolized MODY-3. MODY is a form of diabetes that is characterized by an autosomal dominant mode of inheritance, onset in childhood or early adulthood (usually before 25 years of age), a primary defect in insulin secretion and frequent insulin-independence at the beginning of the disease. Defects in HNF1A are the cause of susceptibility to diabetes mellitus insulin-dependent type 20 (IDDM20). IDDM20 is a multifactorial disorder of glucose homeostasis that is characterized by susceptibility to ketoacidosis in the absence of insulin therapy. Clinical fetaures are polydipsia, polyphagia and polyuria which result from hyperglycemia-induced osmotic diuresis and secondary thirst. These features can result in long-term complications that affect the eyes, kidneys, nerves, and blood vessels. Belongs to the HNF1 homeobox family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Transcription factor; DNA-binding Cellular Component: cytoplasm; nucleus; photoreceptor outer segment; pronucleus; protein complex; transcription factor complex Molecular Function:DNA binding; double-stranded DNA binding; protein binding; protein dimerization activity; protein heterodimerization activity; protein homodimerization activity; sequence-specific DNA binding; transcription factor activity; transcription factor binding Biological Process: bile acid and bile salt transport; bile acid biosynthetic process; blastocyst development; bone resorption; cholesterol metabolic process; chromatin remodeling; embryonic limb morphogenesis; endocrine pancreas development; fatty acid biosynthetic process; fatty acid transport; glucose homeostasis; glucose import; heme biosynthetic process; histone acetylation; insulin secretion; liver development; negative regulation of transcription from RNA polymerase II promoter; paraxial mesoderm formation; placenta development; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; protein localization; regulation of hormone secretion; regulation of insulin secretion; regulation of transcription from RNA polymerase II promoter; regulation of transcription, DNA-dependent; regulation of Wnt receptor signaling pathway; reproductive structure development; response to glucose stimulus; response to oxidative stress; reverse cholesterol transport; transcription from RNA polymerase II promoter; transcription, DNA-dependent
NCBI Summary:	This gene encodes a hepatic transcription factor. The encoded protein is not a member of the T-cell factor family, and is distinct from T-cell specific transcription factor 7 which has also been referred to by the symbol Tcf1. [provided by RefSeq, Jul 2008]
UniProt Code:	P22361
NCBI GenInfo Identifier:	166295202
NCBI Gene ID:	21405
NCBI Accession:	NP_033353.2
UniProt Secondary Accession:	P22361,E9QP86,
UniProt Related Accession:	P22361
Molecular Weight:	67,253 Da
NCBI Full Name:	hepatocyte nuclear factor 1-alpha
NCBI Synonym Full Names:	HNF1 homeobox A
NCBI Official Symbol:	Hnf1a
NCBI Official Synonym Symbols:	HNF1; LFB1; Tcf1; Hnf-1; Tcf-1; HNF1[a]; AI323641; Hnf1alpha; HNF1-alpha
NCBI Protein Information:	hepatocyte nuclear factor 1-alpha
UniProt Protein Name:	Hepatocyte nuclear factor 1-alpha
UniProt Synonym Protein Names:	Liver-specific transcription factor LF-B1; LFB1; Transcription factor 1; TCF-1
Protein Family:	Hepatocyte nuclear factor
UniProt Gene Name:	Hnf1a
UniProt Entry Name:	HNF1A_MOUSE

*ノート: プロトコルは、各バッチ/ロットに固有です。正しい手順については、キットに含まれているプロトコルに従ってください。

ステップ	手順
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.

ELISAアッセイを実施する場合、可能な限り最良の結果を達成するためにサンプルを準備することが重要です。以下に、さまざまなサンプルタイプのサンプルを準備するための手順のリストを示します。

サンプルタイプ	プロトコル
血清:	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.
プラズマ:	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.
尿および脳脊髄液:	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.
細胞培養上清:	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.
細胞溶解物:	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.
組織ホモジネート:	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.
組織溶解物:	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.
母乳:	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.