人間 ZAP70 ELISA Kit (HUEB2540)

商品名:	Human Tyrosine-protein kinase ZAP-70 (ZAP70) ELISA Kit
製品コード:	HUEB2540
エイリアス:	Tyrosine-protein kinase ZAP-70, 70 kDa zeta-associated protein, Syk-related tyrosine kinase, ZAP70, SRK, 2.7.10.2
Uniprot:	P43403
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:	ZAP70: a tyrosine kinase of the Syk family. Associates with the T-cell antigen receptor zeta-chain after TCR stimulation. Phosphorylated by Src-family kinases following antigen receptor activation. Plays a role in lymphocyte activation.
UniProt Protein Details:	Protein type:Kinase, protein; Protein kinase, TK; EC 2.7.10.2; Protein kinase, tyrosine (non-receptor); TK group; Syk family Chromosomal Location of Human Ortholog: 2q12 Cellular Component: cytoplasm; cytosol; extrinsic to internal side of plasma membrane; immunological synapse; intercellular junction; lipid raft; plasma membrane; T cell receptor complex Molecular Function:ATP binding; non-membrane spanning protein tyrosine kinase activity; phosphotyrosine binding; protein binding; protein-tyrosine kinase activity; receptor binding Biological Process: adaptive immune response; B cell activation; B cell receptor signaling pathway; beta selection; immune response; innate immune response; macrophage activation during immune response; negative thymic T cell selection; neutrophil activation during immune response; peptidyl-tyrosine phosphorylation; positive regulation of alpha-beta T cell differentiation; positive regulation of alpha-beta T cell proliferation; positive regulation of B cell differentiation; positive regulation of calcium-mediated signaling; positive regulation of cell adhesion mediated by integrin; positive regulation of mast cell degranulation; positive regulation of T cell differentiation; positive thymic T cell selection; protein amino acid phosphorylation; T cell activation; T cell differentiation; T cell receptor signaling pathway; transmembrane receptor protein tyrosine kinase signaling pathway Disease: Selective T-cell Defect
NCBI Summary:	This gene encodes an enzyme belonging to the protein tyrosine kinase family, and it plays a role in T-cell development and lymphocyte activation. This enzyme, which is phosphorylated on tyrosine residues upon T-cell antigen receptor (TCR) stimulation, functions in the initial step of TCR-mediated signal transduction in combination with the Src family kinases, Lck and Fyn. This enzyme is also essential for thymocyte development. Mutations in this gene cause selective T-cell defect, a severe combined immunodeficiency disease characterized by a selective absence of CD8-positive T-cells. Two transcript variants that encode different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P43403
NCBI GenInfo Identifier:	1177044
NCBI Gene ID:	7535
NCBI Accession:	P43403.1
UniProt Secondary Accession:	P43403,Q6PIA4, Q8IXD6, Q9UBS6, A6NFP4,
UniProt Related Accession:	P43403
Molecular Weight:	55,873 Da
NCBI Full Name:	Tyrosine-protein kinase ZAP-70
NCBI Synonym Full Names:	zeta chain of T cell receptor associated protein kinase 70kDa
NCBI Official Symbol:	ZAP70
NCBI Official Synonym Symbols:	SRK; STD; TZK; STCD; ZAP-70
NCBI Protein Information:	tyrosine-protein kinase ZAP-70
UniProt Protein Name:	Tyrosine-protein kinase ZAP-70
UniProt Synonym Protein Names:	70 kDa zeta-chain associated protein; Syk-related tyrosine kinase
Protein Family:	Tyrosine-protein kinase
UniProt Gene Name:	ZAP70
UniProt Entry Name:	ZAP70_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 ZAP70 ELISA Kit	Anti-ZAP70 Antibody (CAB2195)
ZAP-70 (Phospho-Tyr315) Colorimetric Cell-Based ELISA Kit	Anti-ZAP70 Antibody (CAB9536)
ZAP-70 (Phospho-Tyr292) Colorimetric Cell-Based ELISA Kit	Anti-Phospho-ZAP70-Y319 Antibody (CABP0467)
ZAP-70 (Phospho-Tyr493) Colorimetric Cell-Based ELISA Kit	Anti-Phospho-ZAP70-Y493 Antibody (CABP0468)
	ZAP70 Antibody (PACO03129)