OGT ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level O-Linked-N-Acetylglucosamine Transferase (OGT) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level O-Linked-N-Acetylglucosamine Transferase (OGT) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

HINCUT-1; HRNT1; MRX106; O-GLCNAC; O-GlcNAc transferase p110 subunit; O-GlcNAc transferase subunit p110; OGT1; O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase) ; O-linked N-acetylglucosamine transferase 110 kDa subunit; UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase; UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit; uridinediphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyl transferase; XLID106.

Catalyzes the transfer of a single N-acetylglucosamine from UDP-GlcNAc to a serine or threonine residue in cytoplasmic and nuclear proteins resulting in their modification with a beta-linked N-acetylglucosamine (O-GlcNAc) (PubMed:26678539) . Glycosylates a large and diverse number of proteins including histone H2B, AKT1, EZH2, PFKL, KMT2E/MLL5, MAPT/TAU and HCFC1 (PubMed:26678539) . Can regulate their cellular processes via cross-talk between glycosylation and phosphorylation or by affecting proteolytic processing (PubMed:26678539) . Probably by glycosylating KMT2E/MLL5, stabilizes KMT2E/MLL5 by preventing its ubiquitination (PubMed:26678539) . Involved in insulin resistance in muscle and adipocyte cells via glycosylating insulin signaling components and inhibiting the 'Thr-308' phosphorylation of AKT1, enhancing IRS1 phosphorylation and attenuating insulin signaling. Involved in glycolysis regulation by mediating glycosylation of 6-phosphofructokinase PFKL, inhibiting its activity (PubMed:22923583) . Component of a THAP1/THAP3-HCFC1-OGT complex that is required for the regulation of the transcriptional activity of RRM1. Plays a key role in chromatin structure by mediating O-GlcNAcylation of 'Ser-112' of histone H2B: recruited to CpG-rich transcription start sites of active genes via its interaction with TET proteins (TET1, TET2 or TET3) (PubMed:22121020, PubMed:23353889) . As part of the NSL complex indirectly involved in acetylation of nucleosomal histone H4 on several lysine residues (PubMed:20018852) . O-GlcNAcylation of 'Ser-75' of EZH2 increases its stability, and facilitating the formation of H3K27me3 by the PRC2/EED-EZH2 complex (PubMed:24474760) . Regulates circadian oscillation of the clock genes and glucose homeostasis in the liver. Stabilizes clock proteins ARNTL/BMAL1 and CLOCK through O-glycosylation, which prevents their ubiquitination and subsequent degradation. Promotes the CLOCK-ARNTL/BMAL1-mediated transcription of genes in the negative loop of the circadian clock such as PER1/2 and CRY1/2 (PubMed:12150998, PubMed:18288188, PubMed:19377461, PubMed:19451179, PubMed:20018868, PubMed:20200153, PubMed:21285374, PubMed:15361863) . O-glycosylates HCFC1 and regulates its proteolytic processing and transcriptional activity (PubMed:21285374, PubMed:28584052, PubMed:28302723) .

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 O-Linked-N-Acetylglucosamine Transferase (OGT) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to O-Linked-N-Acetylglucosamine Transferase (OGT) . 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 O-Linked-N-Acetylglucosamine Transferase (OGT), 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 O-Linked-N-Acetylglucosamine Transferase (OGT) 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 (%) _x005F Serum 80-103 98 _x005F Matrix Recovery Range (%) Average (%) Matrix Recovery Range (%) Average (%) Serum 80-103 98 Serum 80-103 98

Component Amount _x005F Anti-OGT Microplate 96 Wells (12 x 8 Well Strips) _x005F OGT Lyophilized Standard 2 x 100 ng _x005F 100X Biotinylated OGT Detector Antibody 120 uL _x005F Avidin/HRP Conjugate 120 uL _x005F Standard Diluent 1 x 20 mL _x005F Detector Antibody Diluent 1 x 12 mL _x005F Conjugate Diluent 1 x 12 mL _x005F 30X Wash Buffer 1 x 20 mL _x005F TMB Substrate 1 x 9 mL _x005F Stop Solution 1 x 6 mL _x005F Component Amount Component Amount Anti-OGT Microplate 96 Wells (12 x 8 Well Strips) Anti-OGT Microplate 96 Wells (12 x 8 Well Strips) OGT Lyophilized Standard 2 x 100 ng OGT Lyophilized Standard 2 x 100 ng 100X Biotinylated OGT Detector Antibody 120 uL 100X Biotinylated OGT 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 O-Linked-N-Acetylglucosamine Transferase (OGT) ._x005F No significant cross-reactivity or interference between O-Linked-N-Acetylglucosamine Transferase (OGT) and analogues was observed.