PADI4 ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Peptidyl Arginine Deiminase Type IV (PADI4) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Peptidyl Arginine Deiminase Type IV (PADI4) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

HL-60 PAD; PAD; PAD4; PADI5; PADI-H protein; PDI4; PDI5; peptidyl arginine deiminase, type IV; peptidyl arginine deiminase, type V; Peptidylarginine deiminase IV; protein-arginine deiminase type IV; protein-arginine deiminase type-4.

Catalyzes the citrullination/deimination of arginine residues of proteins such as histones, thereby playing a key role in histone code and regulation of stem cell maintenance. Citrullinates histone H1 at 'Arg-54' (to form H1R54ci), histone H3 at 'Arg-2', 'Arg-8', 'Arg-17' and/or 'Arg-26' (to form H3R2ci, H3R8ci, H3R17ci, H3R26ci, respectively) and histone H4 at 'Arg-3' (to form H4R3ci) . Acts as a key regulator of stem cell maintenance by mediating citrullination of histone H1: citrullination of 'Arg-54' of histone H1 (H1R54ci) results in H1 displacement from chromatin and global chromatin decondensation, thereby promoting pluripotency and stem cell maintenance. Promotes profound chromatin decondensation during the innate immune response to infection in neutrophils by mediating formation of H1R54ci. Citrullination of histone H3 prevents their methylation by CARM1 and HRMT1L2/PRMT1 and represses transcription. Citrullinates EP300/P300 at 'Arg-2142', which favors its interaction with NCOA2/GRIP1.

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 Peptidyl Arginine Deiminase Type IV (PADI4) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Peptidyl Arginine Deiminase Type IV (PADI4) . 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 Peptidyl Arginine Deiminase Type IV (PADI4), 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 Peptidyl Arginine Deiminase Type IV (PADI4) 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) 82-94 89 EDTA Plasma (n=5) 78-94 89 Heparin Plasma (n=5) 86-95 91 Matrix Recovery Range (%) Average (%) Matrix Recovery Range (%) Average (%) Serum (n=5) 82-94 89 Serum (n=5) 82-94 89 EDTA Plasma (n=5) 78-94 89 EDTA Plasma (n=5) 78-94 89 Heparin Plasma (n=5) 86-95 91 Heparin Plasma (n=5) 86-95 91

Component Amount Anti-PADI4 Microplate 96 Wells (12 x 8 Well Strips) PADI4 Lyophilized Standard 2 x 20 ng 100X Biotinylated PADI4 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-PADI4 Microplate 96 Wells (12 x 8 Well Strips) Anti-PADI4 Microplate 96 Wells (12 x 8 Well Strips) PADI4 Lyophilized Standard 2 x 20 ng PADI4 Lyophilized Standard 2 x 20 ng 100X Biotinylated PADI4 Detector Antibody 120 uL 100X Biotinylated PADI4 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 Peptidyl Arginine Deiminase Type IV (PADI4) . No significant cross-reactivity or interference between Peptidyl Arginine Deiminase Type IV (PADI4) and analogues was observed.