PAK1 Colorimetric Cell-Based ELISA Kit (CBCAB00189)

Product Name:	PAK1 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00189
ELISA Type:	Cell-Based
Target:	PAK1
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The PAK1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PAK1 protein expression profile in cells. The kit can be used for measuring the relative amounts of PAK1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on PAK1.

Qualitative determination of PAK1 concentration is achieved by an indirect ELISA format. In essence, PAK1 is captured by PAK1-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 5058, UniProt ID: Q13153, OMIM: 602590, Unigene: Hs.435714
Gene Symbol:	PAK1
Sub Type:	None

UniProt Protein Function:	PAK1: a protein kinase of the STE20 family that regulates cell motility and morphology. Critical RhoGTPase effector that regulates cytoskeleton reorganization, the JNK MAPK pathway, and nuclear signaling. Binding of Rac/cdc42 to the PBD of PAK1 causes autophosphorylation and conformational change. Phosphorylated and activated by PDK.
UniProt Protein Details:	Protein type:Protein kinase, Ser/Thr (non-receptor); Kinase, protein; EC 2.7.11.1; Protein kinase, STE; STE group; STE20 family; PAKA subfamily Chromosomal Location of Human Ortholog: 11q13-q14 Cellular Component: axon; cytoplasm; cytosol; dendrite; filamentous actin; focal adhesion; Golgi apparatus; growth cone; intercellular junction; nuclear membrane; plasma membrane; protein complex; ruffle; Z disc Molecular Function:ATP binding; collagen binding; protein binding; protein kinase activity; protein kinase binding; protein serine/threonine kinase activity; Rac GTPase binding Biological Process: actin cytoskeleton reorganization; activation of protein kinase activity; amygdala development; apoptosis; axon guidance; branching morphogenesis of a tube; cell migration; cellular response to insulin stimulus; ephrin receptor signaling pathway; exocytosis; innate immune response; MAPKKK cascade; neurite morphogenesis; neuromuscular junction development; positive regulation of cell migration; positive regulation of estrogen receptor signaling pathway; positive regulation of JNK activity; positive regulation of peptidyl-serine phosphorylation; positive regulation of protein amino acid phosphorylation; positive regulation of stress fiber formation; protein amino acid autophosphorylation; protein amino acid phosphorylation; receptor clustering; regulation of apoptosis; regulation of gene expression; regulation of mitotic cell cycle; response to hypoxia; Rho protein signal transduction; small GTPase mediated signal transduction; stimulatory C-type lectin receptor signaling pathway; stress-activated protein kinase signaling pathway; T cell costimulation; T cell receptor signaling pathway; vascular endothelial growth factor receptor signaling pathway; wound healing
NCBI Summary:	This gene encodes a family member of serine/threonine p21-activating kinases, known as PAK proteins. These proteins are critical effectors that link RhoGTPases to cytoskeleton reorganization and nuclear signaling, and they serve as targets for the small GTP binding proteins Cdc42 and Rac. This specific family member regulates cell motility and morphology. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Apr 2010]
UniProt Code:	Q13153
NCBI GenInfo Identifier:	90111767
NCBI Gene ID:	5058
NCBI Accession:	Q13153.2
UniProt Secondary Accession:	Q13153,O75561, Q13567, Q32M53, Q32M54, Q86W79,
UniProt Related Accession:	Q13153
Molecular Weight:
NCBI Full Name:	Serine/threonine-protein kinase PAK 1
NCBI Synonym Full Names:	p21 protein (Cdc42/Rac)-activated kinase 1
NCBI Official Symbol:	PAK1
NCBI Official Synonym Symbols:	PAKalpha
NCBI Protein Information:	serine/threonine-protein kinase PAK 1
UniProt Protein Name:	Serine/threonine-protein kinase PAK 1
UniProt Synonym Protein Names:	Alpha-PAK; p21-activated kinase 1; PAK-1; p65-PAK
Protein Family:	Serine/threonine-protein kinase
UniProt Gene Name:	PAK1
UniProt Entry Name:	PAK1_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-PAK1 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.)