人間 Forkhead box protein O3 (FOXO3) ELISA Kit

商品名:	Human Forkhead box protein O3 (FOXO3) ELISA Kit
製品コード:	HUEB0845
エイリアス:	Forkhead box protein O3, AF6q21 protein, Forkhead in rhabdomyosarcoma-like 1, FOXO3, FKHRL1, FOXO3A
Uniprot:	O43524
反応性:	Human
範囲:	0.312-20 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:	FOXO3A: a transcription factors of the forkhead family. Contains 1 fork-head domain. May regulate apoptosis through control of genes necessary for cell death. Translocation of this gene with the MLL gene is associated with secondary acute leukemia.
UniProt Protein Details:	Protein type:Oncoprotein; DNA-binding; Transcription factor; Autophagy Chromosomal Location of Human Ortholog: 6q21 Cellular Component: nucleoplasm; membrane; cytoplasm; cytosol; nucleus Molecular Function:protein binding; DNA binding; chromatin DNA binding; sequence-specific DNA binding; transcription factor activity; protein kinase binding Biological Process: epidermal growth factor receptor signaling pathway; transcription from RNA polymerase II promoter; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; DNA damage response, signal transduction by p53 class mediator; nerve growth factor receptor signaling pathway; initiation of primordial ovarian follicle growth; positive regulation of transcription, DNA-dependent; antral ovarian follicle growth; positive regulation of erythrocyte differentiation; negative regulation of transcription from RNA polymerase II promoter; glucose homeostasis; ovulation from ovarian follicle; oocyte maturation; regulation of translation; regulation of transcription from RNA polymerase II promoter; positive regulation of neuron apoptosis; innate immune response; positive regulation of transcription from RNA polymerase II promoter
NCBI Summary:	This gene belongs to the forkhead family of transcription factors which are characterized by a distinct forkhead domain. This gene likely functions as a trigger for apoptosis through expression of genes necessary for cell death. Translocation of this gene with the MLL gene is associated with secondary acute leukemia. Alternatively spliced transcript variants encoding the same protein have been observed. [provided by RefSeq, Jul 2008]
UniProt Code:	O43524
NCBI GenInfo Identifier:	8134467
NCBI Gene ID:	2309
NCBI Accession:	O43524.1
UniProt Secondary Accession:	O43524,O15171, Q5T2I7, Q9BZ04, B4DVZ6, E1P5E6,
UniProt Related Accession:	O43524
Molecular Weight:
NCBI Full Name:	Forkhead box protein O3
NCBI Synonym Full Names:	forkhead box O3
NCBI Official Symbol:	FOXO3
NCBI Official Synonym Symbols:	FOXO2; AF6q21; FKHRL1; FOXO3A; FKHRL1P2
NCBI Protein Information:	forkhead box protein O3; forkhead box O3A; forkhead in rhabdomyosarcoma-like 1; forkhead homolog (rhabdomyosarcoma) like 1; forkhead, Drosophila, homolog of, in rhabdomyosarcoma-like 1
UniProt Protein Name:	Forkhead box protein O3
UniProt Synonym Protein Names:	AF6q21 protein; Forkhead in rhabdomyosarcoma-like 1
Protein Family:	Forkhead box protein
UniProt Gene Name:	FOXO3
UniProt Entry Name:	FOXO3_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.

ELISA	Antibodies
Human FOXO3 / Forkhead box protein O3 ELISA Kit	Anti-FOXO3A Antibody (CAB9270)
Human FOXO3 (Forkhead Box Protein O3) CLIA Kit (HUES00648)	Anti-Phospho-FOXO3-S253 Antibody (CABP0684)
Human FOXO3 (Forkhead Box Protein O3) ELISA Kit (HUES02194)	Anti-Phospho-FOXO3-S294 pAb Antibody (CABP0856)
FKHRL1 (Phospho-Ser253) Transcription Factor Activity Assay	Anti-Phospho-FOXO3-S253 Antibody (CABP1131)
	Phospho-FOXO3 (S253) Antibody (PACO02753)