PAK4 ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level p21 Protein Activated Kinase 4 (PAK4) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level p21 Protein Activated Kinase 4 (PAK4) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

P21 protein (Cdc42/Rac) -activated kinase 4; p21 (CDKN1A) -activated kinase 4; p21-activated kinase 4; protein kinase related to S. cerevisiae STE20, effector for Cdc42Hs; serine/threonine-protein kinase PAK 4.

Serine/threonine protein kinase that plays a role in a variety of different signaling pathways including cytoskeleton regulation, cell migration, growth, proliferation or cell survival. Activation by various effectors including growth factor receptors or active CDC42 and RAC1 results in a conformational change and a subsequent autophosphorylation on several serine and/or threonine residues. Phosphorylates and inactivates the protein phosphatase SSH1, leading to increased inhibitory phosphorylation of the actin binding/depolymerizing factor cofilin. Decreased cofilin activity may lead to stabilization of actin filaments. Phosphorylates LIMK1, a kinase that also inhibits the activity of cofilin. Phosphorylates integrin beta5/ITGB5 and thus regulates cell motility. Phosphorylates ARHGEF2 and activates the downstream target RHOA that plays a role in the regulation of assembly of focal adhesions and actin stress fibers. Stimulates cell survival by phosphorylating the BCL2 antagonist of cell death BAD. Alternatively, inhibits apoptosis by preventing caspase-8 binding to death domain receptors in a kinase independent manner. Plays a role in cell-cycle progression by controlling levels of the cell-cycle regulatory protein CDKN1A and by phosphorylating RAN.

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to p21 Protein Activated Kinase 4 (PAK4) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to p21 Protein Activated Kinase 4 (PAK4) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain p21 Protein Activated Kinase 4 (PAK4), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm +/- 10nm. The concentration of p21 Protein Activated Kinase 4 (PAK4) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Matrix Recovery Range (%) Average (%) serum (n=5) 94-103 97 EDTA plasma (n=5) 87-103 99 heparin plasma (n=5) 84-101 93 Matrix Recovery Range (%) Average (%) Matrix Recovery Range (%) Average (%) serum (n=5) 94-103 97 serum (n=5) 94-103 97 EDTA plasma (n=5) 87-103 99 EDTA plasma (n=5) 87-103 99 heparin plasma (n=5) 84-101 93 heparin plasma (n=5) 84-101 93

Component Amount Anti-PAK4 Microplate 96 Wells (12 x 8 Well Strips) PAK4 Lyophilized Standard 2 x 10 ng 100X Biotinylated PAK4 Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Component Amount Component Amount Anti-PAK4 Microplate 96 Wells (12 x 8 Well Strips) Anti-PAK4 Microplate 96 Wells (12 x 8 Well Strips) PAK4 Lyophilized Standard 2 x 10 ng PAK4 Lyophilized Standard 2 x 10 ng 100X Biotinylated PAK4 Detector Antibody 120 uL 100X Biotinylated PAK4 Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL

This assay has high sensitivity and excellent specificity for detection of p21 Protein Activated Kinase 4 (PAK4) . No significant cross-reactivity or interference between p21 Protein Activated Kinase 4 (PAK4) and analogues was observed.