HMGB1 ELISA Kit (Rat)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level High Mobility Group Protein 1 (HMGB1) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level High Mobility Group Protein 1 (HMGB1) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

Ac2-008; Ac2-008-like; amphoterin; heparin-binding protein p30; high mobility group 1; high mobility group protein 1; high mobility group protein B1; Hmg1; HMG-1.

Multifunctional redox sensitive protein with various roles in different cellular compartments. In the nucleus is one of the major chromatin-associated non-histone proteins and acts as a DNA chaperone involved in replication, transcription, chromatin remodeling, V (D) J recombination, DNA repair and genome stability. Proposed to be an universal biosensor for nucleic acids. Promotes host inflammatory response to sterile and infectious signals and is involved in the coordination and integration of innate and adaptive immune responses. In the cytoplasm functions as sensor and/or chaperone for immunogenic nucleic acids implicating the activation of TLR9-mediated immune responses, and mediates autophagy. Acts as danger associated molecular pattern (DAMP) molecule that amplifies immune responses during tissue injury. Released to the extracellular environment can bind DNA, nucleosomes, IL-1 beta, CXCL12, AGER isoform 2/sRAGE, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and activates cells through engagement of multiple surface receptors. In the extracellular compartment fully reduced HMGB1 (released by necrosis) acts as a chemokine, disulfide HMGB1 (actively secreted) as a cytokine, and sulfonyl HMGB1 (released from apoptotic cells) promotes immunological tolerance (PubMed:23519706, PubMed:23446148, PubMed:23994764, PubMed:25048472) . Has proangiogenic activity. May be involved in platelet activation. Binds to phosphatidylserine and phosphatidylethanolamide (PubMed:11154118) . Bound to RAGE mediates signaling for neuronal outgrowth (PubMed:1885601, PubMed:2461949, PubMed:7592757, PubMed:12183440) . May play a role in accumulation of expanded polyglutamine (polyQ) proteins.

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 High Mobility Group Protein 1 (HMGB1) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to High Mobility Group Protein 1 (HMGB1) . 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 High Mobility Group Protein 1 (HMGB1), 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 High Mobility Group Protein 1 (HMGB1) 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) 83-103 91 EDTA Plasma (n=5) 96-103 101 Heparin Plasma (n=5) 79-99 87 Matrix Recovery Range (%) Average (%) Matrix Recovery Range (%) Average (%) Serum (n=5) 83-103 91 Serum (n=5) 83-103 91 EDTA Plasma (n=5) 96-103 101 EDTA Plasma (n=5) 96-103 101 Heparin Plasma (n=5) 79-99 87 Heparin Plasma (n=5) 79-99 87

Component Amount HMGB1 Microplate 96 Wells (12 x 8 Well strips) HMGB1 Lyophilized Standard 2 x 3 ng 100X Biotinylated HMGB1 Detector Antibody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 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 Stop Solution 1 x 6 mL TMB Substrate 1 x 9 mL Component Amount Component Amount HMGB1 Microplate 96 Wells (12 x 8 Well strips) HMGB1 Microplate 96 Wells (12 x 8 Well strips) HMGB1 Lyophilized Standard 2 x 3 ng HMGB1 Lyophilized Standard 2 x 3 ng 100X Biotinylated HMGB1 Detector Antibody 1 x 120 uL 100X Biotinylated HMGB1 Detector Antibody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 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 Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL