| Description | Products contentS665634Component1 mL5 mLStorageS665634A2×Super Pfx Master Mix1 mL 5×1 mL-20℃. Avoid freeze/thaw cycle.S665634BddH₂O1 mL 5×1 mL-20℃. Avoid freeze/thaw cycle. Products IntroductionIt is an optimized 2× premixed reagent consisting of Super Pfx DNA Products contentS665634Component1 mL5 mLStorageS665634A2×Super Pfx Master Mix1 mL 5×1 mL-20℃. Avoid freeze/thaw cycle.S665634BddH₂O1 mL 5×1 mL-20℃. Avoid freeze/thaw cycle. Products IntroductionIt is an optimized 2× premixed reagent consisting of Super Pfx DNA Polymerase, Mg2+, dNTPs, and reaction buffer, and requires only the addition of primers and DNA templates for the PCR reaction.Super Pfx DNA Polymerase has a 3´→5´ exonuclease activity, and is approximately 100 times more fidelity than Taq DNA Polymerase. Super Pfx DNA Polymerase has 3´→5´ exonuclease activity and is about 100 times more fidelity than Taq DNA Polymerase, making it ideal for cloning. With the introduction of elongation enhancement technology, this enzyme enables high-speed PCR with an elongation speed of 10 sec/kb and an amplification length of up to 20 kb, while maintaining high fidelity. In addition, this product combines a high success rate with the ability to amplify long fragments that are difficult to amplify, GC-rich regions, and templates that are too large to be amplified. The amplified PCR product does not have an "A" base at the 3´ end and can be directly cloned into a flat-end vector. This product is divided into the standard 2×Master Mix without Loading Dye and the 2×Master Mix with Loading Dye.Product Characteristics1. High-speed PCR possibleThe extension time can be set to 10sec for amplification of target fragments of less than 1kb, and 20-30sec/kb for amplification of target fragments of 1-10kb, which significantly reduces the PCR reaction time compared to conventional reagents.2. High fidelityThe fidelity of Taq DNA polymerase is about 100 times higher than that of Taq DNA polymerase, and long target fragments can be amplified quickly and with high fidelity, and the amplified products can be used for a variety of purposes.3. simple and convenientThis reagent contains all PCR components except primers and templates, which facilitates the operation and improves the reproducibility of the results. In addition, 2×Super Pfx Master Mix (Dye) contains Loading Dye, which can be used for gel electrophoresis directly after the reaction.caveatPrimers and templates with uracil are not suitable for this product.Instructions for usePCR Reactors1. Before preparing the reaction solution, please melt and mix the reagents completely before use.After adding the components, please mix them well and transfer them to the PCR instrument quickly.・The recommended final primer concentration is 0.4-0.5 µM, but for amplification of long fragments of 10 kb or more, a final primer concentration of 0.3 µM can increase the amount of amplified product.PCR reaction program1. Before preparing the reaction solution, please melt and mix the reagents completely before use.take note of 1）Pre-denaturation: for most purified templates, 98℃, 30sec is sufficient; for complex templates, the pre-denaturation time can be extended, not more than 3min. 2）Annealing: In general, the annealing temperature is 3-5°C lower than the primer Tm. When a non-specific reaction occurs, increase the annealing temperature appropriately. If necessary, a temperature gradient can be established to find the optimal temperature for primer-template binding. For high Tm primers, a two-step cycle can be used, combining annealing and extension into one step.3）Extension: for complex genomic samples, extension times are typically 20-30sec/kb, but can be reduced to 10sec/kb for simple templates (plasmids, E. coli, etc.) or complex templates <1kb. cDNAs, or complex templates >6kb, can be increased to 40-50sec/kb, if desired... Read More | General DescriptionNatural human C5a is prepared from human C5 protein cleaved into C5a and C5b by human C5 convertase. The C5a is converted to C5a desArg by proteolytic removal of the C-terminal arginine. The primary carboxypeptidase responsible for Arg removal is serum carboxypeptidase N, but General DescriptionNatural human C5a is prepared from human C5 protein cleaved into C5a and C5b by human C5 convertase. The C5a is converted to C5a desArg by proteolytic removal of the C-terminal arginine. The primary carboxypeptidase responsible for Arg removal is serum carboxypeptidase N, but there are several different carboxypepticases in serum. C5a desArg is a naturally glycosylated polypeptide containing 73 amino acids with a molecular weight of approx. 10,250 daltons. It contains 25% carbohydrate attached to a single Asn residue at position 64. This carbohydrate is of variable structure leading to a broad distribution of MW upon analysis by mass spectroscopy. C5a is the most potent anaplylatoxin (compared to C3a and C4a). C5a desArg is produced when C5a is“inactivated” by removal of its C-terminal arginine amino acid. This cleavage occurs by the action of the plasma enzyme carboxypeptidase N. This inactivation is rapid and most C5a is converted to C5a desArg within minutes of its formation. “Inactivated” C5a still possesses approx. 1% of its anaphylatoxic and chemotatic activities, but its stimulatory activity is only reduced 10-fold. Thus, C5a desArg retains considerable biological activity even though it is frequently called inactivated C5a. Its biological properties include being weakly chemotactic for neutrophils (PMN), causing smooth muscle contraction, increasing vascular permeability, causing histamine and TNF-alpha release, and causing lysosomal degranulation of immune cells. C5a and C5a desArg act through the C5a Receptor (C5aR, CD88, a G-protein coupled receptor) on PMN, monocytes, alveolar macrophages, and mast cells. A second receptor of unknown function (C5L2, gpr77) has been identified. Due to the widespread expression of C5a receptors and the results from C5aR KO mice it is believed that C5a and its receptors have many nonimmunolgical functions in organ development, CNS development, neurodegeneration, tissue regeneration and hematopoiesis (Monk, P.N. et al. (2007)).Native versus Recombinant C5a desArgNumerous recombinant forms of C5a and C5a desArg are sold by many companies. In side-by-side biological testing, we have found that our native native proteins are 10- to 100-fold more active per µg than all but one of these recombinant proteins. Structurally not a single one of the recombinant proteins on the market has the correct amino acid sequence or structure. They have extra amino acids at the N-terminal (such as 6 His tags), different amino acids in the sequence itself (some were produced from the original, but incorrect amino acid sequence), and none possess the 25% carbohydrate at Asn 64. In fact, one recombinant C5a on the market has approximately 30 additional amino acids at the N-terminal end due to the cloning vector used. This is a 40% addition of nonsense structure to the C5a molecule. Both our C5a and our C5adesArg are native proteins produced by the native human C5 convertase.Physical Characteristics & StructureDeglycosylated MW: Calculated monoisotopic mass 8112; Calculated average mass 8117.Isoelectric point: pI = 8.8Carbohydrate content: ~25% carbohydrate (heterogeneous) Amino acid sequence: TLQKKIEEIA AKYKHSVVKK CCYDGACVNN DETCEQRAAR ISLGPRCIKA FTECCVVASQ LRANISHKDM QLGMDL Number: MFCD00130842NMRderived structure: FEBS Lett. 238:289-294, 1988; Biochemistry 28:172-185,1989; Biochemistry 29:2895-2905, 1990; Proteins 28:261-267, 1997.Extinction Coeff. A280 nm = 0.41 at 1.0 mg/mlPurity: > 97% by SDS-PAGEAssaysThe multitude of biological functions of C5a has resulted in the use of many different assay systems. The most typical biological assays being smooth muscle contraction assays using guinea pig ileum, chemotaxis assays using neutrophils or granule-release assays using human PMN or similar cell lines. Granule release is generally followed by measuring the release of myeloperoxidase. Functional responses have been detected in the picomolar concentration range (Gerard, C. et al. (1981); Hugli, T.E. et al. (1981)).ELISA kits for the assay of C5a and C5a desArg in blood and other fluids are sold by many companies. These measurements are useful for detecting complement activation in vivo, but the interpretation of their meaning is complicated by the fact that clearance of the anaphylatoxins is rapid.In vivoThe resting serum concentration of C5a desArg has been reported to be approximately 4 nM although it is difficult to draw, store and test blood without 1 to 10 % C5 activation (Watkins, J. (1987)). The presence of EDTA and Futhan in the collection tubes can minimize this background. Full activation of all C5 in blood (75 µg/mL) would result in ~380 nM C5a (~3.9 µg/mL). Due to the extreme sensitivity of many C5a responses, a response can theoretically be initiated by activation of approximately one millionth of the C5 in a local area (sub-picomolar C5a).RegulationC5adesArg levels are regulated by two processes: formation and clearance. The enzymes that cleave C5 and release C5a (collectively called C5 convertases) do so at very slow rates. Operating at Vmax the best enzymes only cleave one C5 every three minutes (Rawal, N. and Pangburn, M.K. (2001)). C5a desArg is created when C5a is“inactivated” by removal of its C-terminal arginine amino acid. The product C5a desArg is produced by the action of the plasma enzyme carboxypeptidase N. This inactivation is rapid and most C5a is converted to C5a desArg within minutes of its formation. “Inactivated” C5a still possesses approx. 1% of its anaphylatoxic and chemotatic activities, but its stimulatory activity is only reduced 10-fold. Thus, C5a desArg retains considerable biological activity even though it is frequently called inactivated C5a. Because of the large number of cells bearing C5a receptors (endothelial, immune, smooth muscle, neuronal, etc.) the capture, internalization and digestion of C5a and C5a desArg results in their rapid removal from circulation.DeficienciesA deficiency of C5 or a deficiency of the enzymes that cleave C5 to generate C5a would result in the absence of C5a and C5a desArg. A knock-out mouse deficient in carboxypeptidase N has been created and found to be hypersensitive to complement activation and CVF administration (Mueller-Ortiz S.L. et al. (2009)). Administration of human C5a was 100% lethal in these KO mice probably due to their inability to inactivate C5a to C5a desArg. There are no known complete deficiencies of C5 convertases. Examples of C5 deficient humans and mice exist. In fact, many laboratory mouse strains in common use were shown to have been bred with a deficiency of C5 (A/HeJ, AKR/J, DBA/2J, NZB/B1NJ, SWR/J, and B10.D2/nSnJ). The lack of C5 prevents formation of the membrane attack complex of complement and precludes formation of C5a and C5a desArg. Humans lacking C5 are susceptible to repeated infections from a wide variety of organisms, primarily gram-negative bacteria. Meningococcal and gonococcal neisserial infections are especially problematic. The degree to which pathologies associated with C5 deficiency are due to the lack of C5 or due to the absence of C5a and C5a desArg is unclear but information on this isbeing acquired from receptor knock-out animals.DiseasesSee Deficiencies above.Precautions/Toxicity/HazardsThis protein is purified from human serum and therefore precautions appropriate for handling any blood-derived product must be used even though the source was shown by certified tests to be negative for HBsAg, HTLV-I/II, STS, and for antibodies to HCV, HIV-1 and HIV-II.Injection can cause anaphylatic shock which is a generalized circulatory collapse similar to that caused by an allergic reaction.Hazard Code: B WGK Germany 3MSDS available upon request... 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 | Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue StainingDescription:NG2, also known as CSPG4, MCSP, and AN2, is a 400-500 kDa transmembrane chondroitin sulfate proteoglycan (CSPG) with a protein core of approximately 300 kDa. The extracellular region can be proteolytically shed fromPurity:>95%, by SDS-PAGE visualized with Coomassie® Blue StainingDescription:NG2, also known as CSPG4, MCSP, and AN2, is a 400-500 kDa transmembrane chondroitin sulfate proteoglycan (CSPG) with a protein core of approximately 300 kDa. The extracellular region can be proteolytically shed from the cell surface. Mature human NG2 consists of a 2195 amino acid (aa) extracellular domain (ECD), a 21 aa transmembrane segment, and a 77 aa cytoplasmic domain. Within aa 1583-2224, human NG2/CSPG4 shares 83% aa sequence identity with mouse and rat CSPG4. NG2 binds to the extracellular matrix proteins Laminin, Tenascin, and Collagens II, V, and VI as well as to the growth factors FGF-2 and PDGF-AA. NG2 is expressed on glial cell progenitors known as O2A cells or NG2 glia. These cells are neuronally responsive and differentiate primarily into oligodendrocytes but also into astrocytes. NG2 associates with PDGF R alpha and the AMPA R subunit GluR2. It is up-regulated on microglial cells during inflammation and contributes to the induction of inflammatory mediators. Various CSPGs in the brain inhibit neurite outgrowth through interactions with Nogo Receptor/NgR1 and NgR3. This recombinant protein product corresponds to the last 5 CSPG repeats, a region which can independently inhibit neurite outgrowth. NG2 is also expressed on vascular mural cells and capillaries. It promotes vascular endothelial cell (EC) migration and angiogenesis through interactions with Galectin-3 and Integrin alpha 3 beta 1 on EC, Plasminogen, and Angiostatin. NG2 is also expressed on a variety of tumors where it contributes to tumor cell adhesion, motility, and invasion... Read More | Inquire |