PAK3 (Phospho-Ser154) Colorimetric Cell-Based ELISA Kit

Product Name:	PAK3 (Phospho-Ser154) Colorimetric Cell-Based ELISA
Product Code:	CBCAB01653
ELISA Type:	Cell-Based
Target:	PAK3 (Phospho-Ser154)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The PAK3 (Phospho-Ser154) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PAK3 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PAK3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on PAK3 phosphorylation.

Qualitative determination of PAK3 (Phospho-Ser154) concentration is achieved by an indirect ELISA format. In essence, PAK3 (Phospho-Ser154) is captured by PAK3 (Phospho-Ser154)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 5063, UniProt ID: O75914, OMIM: 300142/300558, Unigene: Hs.656789
Gene Symbol:	PAK3
Sub Type:	Phospho

UniProt Protein Function:	PAK3: a protein kinase of the STE20 family that regulates synapse formation and plasticity in the hippocampus. May be necessary for dendritic development and for the rapid cytoskeletal reorganization in dendritic spines associated with synaptic plasticity. Forms an activated complex with GTP-bound P21, CDC2 and RAC1 proteins. Missense and truncation mutations linked to nonsyndromic mental retardation type 30 (MRX30). Two alternatively spliced human isoforms have been reported.
UniProt Protein Details:	Protein type:Protein kinase, STE; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; EC 2.7.11.1; STE group; STE20 family; PAKA subfamily Chromosomal Location of Human Ortholog: Xq23 Cellular Component: cytoplasm; cytosol; endosome; plasma membrane Molecular Function:ATP binding; MAP kinase kinase activity; metal ion binding; protein binding; protein serine/threonine kinase activity; Rac GTPase binding; SH3 domain binding Biological Process: actin cytoskeleton organization and biogenesis; activation of MAPK activity; axon guidance; axonogenesis; cell migration; dendrite development; ephrin receptor signaling pathway; innate immune response; MAPKKK cascade; positive regulation of neuron apoptosis; regulation of actin filament polymerization; regulation of apoptosis; regulation of mitotic cell cycle; Rho protein signal transduction; small GTPase mediated signal transduction; stimulatory C-type lectin receptor signaling pathway; stress-activated protein kinase signaling pathway; synapse organization and biogenesis; T cell costimulation; T cell receptor signaling pathway; vascular endothelial growth factor receptor signaling pathway Disease: Mental Retardation, X-linked 30
NCBI Summary:	PAK proteins are critical effectors that link Rho GTPases to cytoskeleton reorganization and nuclear signaling. PAK proteins, a family of serine/threonine p21-activating kinases, serve as targets for the small GTP binding proteins Cdc42 and RAC and have been implicated in a wide range of biological activities. The protein encoded by this gene forms an activated complex with GTP-bound RAS-like (P21), CDC2 and RAC1 proteins which then catalyzes a variety of targets. This protein may be necessary for dendritic development and for the rapid cytoskeletal reorganization in dendritic spines associated with synaptic plasticity. Defects in this gene are the cause of non-syndromic mental retardation X-linked type 30 (MRX30), also called X-linked mental retardation type 47 (MRX47). Alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Jul 2008]
UniProt Code:	O75914
NCBI GenInfo Identifier:	47117818
NCBI Gene ID:	5063
NCBI Accession:	O75914.2
UniProt Secondary Accession:	O75914,Q5JWX1, Q5JWX2, Q7Z2D6, Q7Z2E4, Q7Z3Z8, Q8WWK5 Q8WX23, A8K389, B1GX77, B1GX78, B1GX79,
UniProt Related Accession:	O75914
Molecular Weight:	62,913 Da
NCBI Full Name:	Serine/threonine-protein kinase PAK 3
NCBI Synonym Full Names:	p21 protein (Cdc42/Rac)-activated kinase 3
NCBI Official Symbol:	PAK3
NCBI Official Synonym Symbols:	bPAK; MRX30; MRX47; OPHN3; PAK-3; PAK3beta; beta-PAK
NCBI Protein Information:	serine/threonine-protein kinase PAK 3
UniProt Protein Name:	Serine/threonine-protein kinase PAK 3
UniProt Synonym Protein Names:	Beta-PAK; Oligophrenin-3; p21-activated kinase 3; PAK-3
Protein Family:	Serine/threonine-protein kinase
UniProt Gene Name:	PAK3
UniProt Entry Name:	PAK3_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.	Incubate the cells for overnight at 37 °C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-PAK3 (Phospho-Ser154) Antibody, Anti-PAK3 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

슈반 세포: 신경 기능을 지원하는 특수 세포

신경면역학: CNS의 면역체계

바르덴부르크 증후군 및 클라인-바르덴부르크 증후군

PAK3 (Phospho-Ser154) Colorimetric Cell-Based ELISA Kit

Description