| Description | Product InfoAssays employing crude extracts for topo II activity based upon relaxation of supercoiled DNA can be complicated due to the presence of topo I in partially purified fractions. Additional complications arise with contaminating nuclease activity (due to Mg++) which degrade or nick the Product InfoAssays employing crude extracts for topo II activity based upon relaxation of supercoiled DNA can be complicated due to the presence of topo I in partially purified fractions. Additional complications arise with contaminating nuclease activity (due to Mg++) which degrade or nick the supercoiled substrate. These problems can be avoided by using a catenated DNA substrate prepared from the kinetoplast of the insect trypanosome Crithidia fasciculata. Kinetoplast DNA (kDNA) is an aggregate of interlocked DNA minicircles (mostly 2.5 kb) that form extremely large networks of high molecular weight. As a result, these networks fail to enter an agarose gel. Upon incubation with topo II, which engages DNA in a double stranded breaking and reunion cycle, minicircular DNAs are effectively released (decatenated). The decatenated minicircles move rapidly into the gel owing to their small size. This reaction will not occur with topoisomerase I. The products of the reaction can vary as follows:Quality Control Tests:For catenated KDNA substrate, at least 90% of the DNA will be retained in the well of a 1% agarose gel.Decatenation of each batch of Kinetoplast DNA is tested with purified topoisomerase II.Storage Buffer:kDNA is stored in 10 mM Tris-Cl (pH 7.5), and 1 mM EDTA at the concentration specified with the product.DescriptionkDNA is the ideal substrate for topoisomerase II assays because it is specific for type II reaction mechanisms. Researchers can even assay for a type II enzyme in the presence of large excess of topoisomerase I. Thus, kDNA works well to quantify type II activity in crude cell extracts, which are frequently overloaded with topoisomerase I. The kDNA substrate works exceptionally well with human Top 2a and b as well as prokaryotic enzymes, such as DNA gyrase or topoisomerase IV. Shipping&storagekDNA should be stored at 4°C; however, long term storage at -20°C is acceptable. The DNA is usually shipped at ambient temperature... Read More | Malic Dehydrogenase is a ubiquitous enzyme, which exists in two isoforms in eukaryotic cells.Malic dehydrogenase exists as a dimer with each subunit containing an NAD-binding domain and a substrate-binding carboxy-terminal domain required for activity. Malic dehydrogenase is a cytoplasmic isozyme Malic Dehydrogenase is a ubiquitous enzyme, which exists in two isoforms in eukaryotic cells.Malic dehydrogenase exists as a dimer with each subunit containing an NAD-binding domain and a substrate-binding carboxy-terminal domain required for activity. Malic dehydrogenase is a cytoplasmic isozyme and an important catalyst in the tricarboxylic acid cycle.ReagentsA. 0.1 M Tris-HCl buffer (pH7.8)B. 0.01 M Phosphate buffer (KH2PO4-NaOH, pH 7.0)C. Triton X-100 solution (50 mg/ml)D. 0.01 M Phosphate buffer containing 0.1% Triton X-100 (KH2PO4-NaOH, pH 7.0)Dilute 20 ml of Triton X-100 solution (C) with approx. 800 ml of 0.01M Phosphate buffer (B). Fill up to 1,000 ml with 0.01M Phosphate buffer (B).E. NADH soluton Weigh 9 mg of NADH and dissolve in 0.1M Tris-HCl bufer (A). Fill up to 50 ml with 0.1M Tris-HCl Buffer (A). (Can be used for 5 days if kept refrigerated)F. Substrate solutionWeigh 11 mg of oxaloacetic acid and dissolve in 0.1M Tris-HCl buffer (A). Fill up to 50 ml with 0.1M Tris-HCl buffer (A) (Make a fresh solution for each use.)G. Enzyme solutionWeigh out Malate Dehydrogenase and dissolve in chilled 0.01M Phosphate Bufer containing 0.1% Triton X-100 (D). Enzyme solution should be prepared so that the value of AOD/minute becomes in the range of 0.025 ± 0.010.ProcedurePipette 2.0 ml of NADH solution (E) and 0.90 ml of Substrate solution (F) respectively into a quartz cell (d=10 mm) and keep at 25 + 0.5'℃ for 5 minutes. Then, pipete 0.10 ml of Enzyme solution (G) into the quartz cell and mix well immediately. Keep the reaction mixture at 25 ±0.5'C.Exaclly at 2 minutes and 5 minutes after the addition of Enzyme solution (G), measure the absorbances of the reaction mixture at 340 nm(A2 and A5).As a blank, pipette 0.01M Phosphate buffer (D) into another quartz cel (d=10 mm) instead of the Enzyme solution (G) and follow the same procedure described above (Ab2 and Ab5).CalculationMalate dehydrogenase activity (u/mg)=[(A2-A5)-(Ab2-Ab5)]/3*(1/6.22)*(n/0.1) ApplicationThis enzyme is used for the enzymatic determination of L-malate and gluamate oxalo-acetate transaminase(GOT)in clinical diagnosis... Read More | Purity:>98%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：Heme oxygenase (HMOX) is the rate limiting enzyme in heme catabolism. It cleaves heme to biliverdin, carbon monoxide, and iron. The biliverdin is subsequently converted to bilirubin by biliverdin reductase. Purity:>98%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：Heme oxygenase (HMOX) is the rate limiting enzyme in heme catabolism. It cleaves heme to biliverdin, carbon monoxide, and iron. The biliverdin is subsequently converted to bilirubin by biliverdin reductase. The mechanism of HMOX is unique in that heme serves as the substrate of the enzyme and as the prosthetic group for the activation of iron-bound O2. HMOX activity is highest in spleen where senescent erythrocytes are sequestered and destroyed. Two isoforms, HMOX1 and HMOX2, are expressed in most tissues. HMOX1 is an inducible enzyme in response to heme, heavy metals, oxidative stress, cytokines, and many drugs. Whereas HMOX2 displays a constitutive expression. HMOX1 is expressed mainly in spleen, liver, and kidney, and HMOX2 is prominently expressed in the brain and testes. The increased expression of HMOX1 levels is related to a variety of pathological states, where it functions as a cytoprotective molecule through its by products. HMOX1 also plays important roles in the regulation of cell proliferation, differentiation, and apoptosis... Read More | Inquire | Format:1-ComponentEnzyme:Horseradish peroxidase |