人間 GSK3B ELISA Kit (HUEB0312)

商品名:	Human Glycogen synthase kinase-3 beta (GSK3B) ELISA Kit
製品コード:	HUEB0312
エイリアス:	Glycogen synthase kinase-3 beta, GSK-3 beta, Serine/threonine-protein kinase GSK3B, GSK3B, 2.7.11.26, 2.7.11.1
Uniprot:	P49841
反応性:	Human
範囲:	0.156-10 ng/mL
検出方法:	Sandwich
サイズ:	96 Assay
保管所:	Please see kit components below for exact storage details
ノート:	For research use only

UniProt Protein Function:	GSK3B: a proline-directed protein kinase of the GSK family. Phosphorylates and inactivates glycogen synthase. Participates in the Wnt signaling pathway. Involved in energy metabolism, neuronal cell development, and body pattern formation
UniProt Protein Details:	Protein type:Protein kinase, CMGC; EC 2.7.11.26; EC 2.7.11.1; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; CMGC group; GSK family; GSK subfamily Chromosomal Location of Human Ortholog: 3q13.3 Cellular Component: axon; beta-catenin destruction complex; cell soma; centrosome; cytoplasm; cytosol; dendritic shaft; dendritic spine; growth cone; lipid raft; mitochondrion; nucleus; perinuclear region of cytoplasm; plasma membrane; ribonucleoprotein complex Molecular Function:ATP binding; beta-catenin binding; integrin binding; ionotropic glutamate receptor binding; kinase activity; NF-kappaB binding; p53 binding; protein binding; protein kinase activity; protein kinase binding; protein serine/threonine kinase activity; tau protein binding; tau-protein kinase activity; ubiquitin protein ligase binding Biological Process: aging; axonogenesis; cell migration; circadian rhythm; dopamine receptor signaling pathway; epithelial to mesenchymal transition; ER overload response; establishment of cell polarity; fat cell differentiation; genetic imprinting; glycogen metabolic process; hippocampus development; myoblast fusion; negative regulation of apoptosis; negative regulation of dendrite morphogenesis; negative regulation of glycogen biosynthetic process; negative regulation of MAP kinase activity; negative regulation of neuron maturation; negative regulation of NFAT protein import into nucleus; negative regulation of nitric-oxide synthase activity; negative regulation of protein binding; negative regulation of protein complex assembly; organ morphogenesis; peptidyl-serine phosphorylation; peptidyl-threonine phosphorylation; positive regulation of axon extension; positive regulation of cell-matrix adhesion; positive regulation of GTPase activity; positive regulation of neuron apoptosis; positive regulation of peptidyl-serine phosphorylation; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; positive regulation of protein binding; positive regulation of protein catabolic process; positive regulation of protein complex assembly; positive regulation of protein export from nucleus; positive regulation of smooth muscle cell proliferation; positive regulation of transcription from RNA polymerase II promoter; proteasomal ubiquitin-dependent protein catabolic process; protein amino acid autophosphorylation; protein amino acid phosphorylation; protein export from nucleus; re-entry into mitotic cell cycle; regulation of microtubule-based process; regulation of neuronal synaptic plasticity; response to activity; response to drug; response to estradiol stimulus; response to insulin stimulus; Wnt receptor signaling pathway; Wnt receptor signaling pathway through beta-catenin
NCBI Summary:	The protein encoded by this gene is a serine-threonine kinase, belonging to the glycogen synthase kinase subfamily. It is involved in energy metabolism, neuronal cell development, and body pattern formation. Polymorphisms in this gene have been implicated in modifying risk of Parkinson disease, and studies in mice show that overexpression of this gene may be relevant to the pathogenesis of Alzheimer disease. Alternatively spliced transcript variants encoding different isoforms have been found for this gene.[provided by RefSeq, Sep 2009]
UniProt Code:	P49841
NCBI GenInfo Identifier:	20455502
NCBI Gene ID:	2932
NCBI Accession:	P49841.2
UniProt Secondary Accession:	P49841,Q9BWH3, Q9UL47, D3DN89,
UniProt Related Accession:	P49841
Molecular Weight:	48,034 Da
NCBI Full Name:	Glycogen synthase kinase-3 beta
NCBI Synonym Full Names:	glycogen synthase kinase 3 beta
NCBI Official Symbol:	GSK3B
NCBI Protein Information:	glycogen synthase kinase-3 beta
UniProt Protein Name:	Glycogen synthase kinase-3 beta
UniProt Synonym Protein Names:	Serine/threonine-protein kinase GSK3B (EC:2.7.11.1)
Protein Family:	Glycogen synthase kinase
UniProt Gene Name:	GSK3B
UniProt Entry Name:	GSK3B_HUMAN

成分	額	保管所
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	-

その他の必要な材料と設備：

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

*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。

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.

ステップ	プロトコル
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.

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.

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