ねずみ GCK/ Glucokinase ELISA Kit

商品名:	Mouse GCK / Glucokinase ELISA Kit
製品コード:	MOFI00271
サイズ:	96 Assays
エイリアス:	Gck, Glucokinase, FGQTL3, GLK, Hexokinase-4, Hexokinase-D, HHF3, HK4, HXKP, LGLK, GK, glucokinase, hexokinase 4, HKIV, maturity onset diabetes of the young 2, MODY2
検出方法:	Sandwich ELISA
申し込み:	This immunoassay kit allows for the in vitro quantitative determination of Mouse Gck concentrations in serum plasma and other biological fluids.
感度:	0.094ng/ml
範囲:	0.156-10ng/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 Gck and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse Gck in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-99	94
EDTA plasma(n=5)	92-105	99
UFH plasma(n=5)	86-103	93

直線性:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse Gck 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)	91-103%	85-104%	92-105%
EDTA plasma(n=5)	92-100%	84-98%	83-99%
UFH plasma(n=5)	80-94%	81-90%	81-100%

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

P52792

UniProt Protein Function:	GK: a glycolytic enzyme that catalyzes glucose metabolism in the liver and pancreatic beta cells. Acts as a ""glucose sensor"" in beta cells. The first and rate-limiting step in glycosis, a pathway that produces energy in the form of ATP from glucose. Glucokinase traps glucose inside the cell by catalyzing its phosphorylation to produce glucose-6-phosphate. Has a lower affinity for glucose than the three other isozymes of hexokinase, allowing other organs such as the brain and muscles to have first call on glucose when its supply is limited. Unlike other hexokinases, glucokinase is not inhibited by glucose-6-phosphate. Glucokinase is found in the outer membrane compartment of mitochondria. May bind VDAC, suppressing mitochondrial function. Glucokinase transcription is induced by insulin, perhaps via the activation of Stat 5B. Mutant glucokinase causes a rare form of diabetes and may also play a role in type 2 diabetes. Three splice variant isoforms of human glucokinase have been described.
UniProt Protein Details:	Protein type:Carbohydrate Metabolism - amino sugar and nucleotide sugar; Kinase, other; Carbohydrate Metabolism - galactose; Mitochondrial; Carbohydrate Metabolism - glycolysis and gluconeogenesis; EC 2.7.1.2; Carbohydrate Metabolism - starch and sucrose Cellular Component: nucleoplasm; mitochondrion; cell; cytoplasm; actin filament; cell cortex; cytosol; basal cortex; nucleus; secretory granule Molecular Function:transferase activity; hexokinase activity; protein binding; magnesium ion binding; nucleotide binding; kinase activity; ADP binding; phosphotransferase activity, alcohol group as acceptor; glucokinase activity; protein phosphatase binding; ATP binding; glucose binding Biological Process: positive regulation of glycogen biosynthetic process; glucose 6-phosphate metabolic process; lipid homeostasis; glucose homeostasis; NADP metabolic process; cellular response to glucose starvation; elevation of cytosolic calcium ion concentration; detection of glucose; negative regulation of gluconeogenesis; second-messenger-mediated signaling; glycogen biosynthetic process; glycolysis; regulation of potassium ion transport; glucose metabolic process; positive regulation of insulin secretion; fructose 2,6-bisphosphate metabolic process; cellular response to insulin stimulus; carbohydrate metabolic process; carbohydrate phosphorylation; positive regulation of glycolysis; negative regulation of epinephrine secretion; cell glucose homeostasis; regulation of insulin secretion; phosphorylation; positive regulation of phosphorylation
UniProt Code:	P52792
NCBI GenInfo Identifier:	31982798
NCBI Gene ID:	103988
NCBI Accession:	NP_034422.2
UniProt Secondary Accession:	P52792,P52791,
UniProt Related Accession:	P52792
Molecular Weight:	52,089 Da
NCBI Full Name:	glucokinase
NCBI Synonym Full Names:	glucokinase
NCBI Official Symbol:	Gck
NCBI Official Synonym Symbols:	Gk; GLK; HK4; HKIV; HXKP; MODY2; Gls006
NCBI Protein Information:	glucokinase; HK IV; hexokinase 4; hexokinase-4; hexokinase-D; hexokinase type IV
UniProt Protein Name:	Glucokinase
UniProt Synonym Protein Names:	Hexokinase type IV; HK IV; Hexokinase-4; HK4; Hexokinase-D
Protein Family:	Glucokinase
UniProt Gene Name:	Gck
UniProt Entry Name:	HXK4_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.