| Description | Product DescriptionEndo H, endo-beta-N-acetylglucosaminidase H, Endoglycosidase HEndo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, Product DescriptionEndo H, endo-beta-N-acetylglucosaminidase H, Endoglycosidase HEndo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.Recommended Reagentsincluded with:1 vial: 5x Reaction Buffer - 400 ml250 mM sodium phosphate, pH5.51 vial: Denaturation Solution - 200 ml 2%SDS, 1 M Beta-mercaptoethanol Molecular weightapproximately 29 kDpH optimum:5.5, active over the range 5-6.ApplicationsReleases asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. Endo H cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosac-charide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.Specific ActivityOne unit of Endo H activity is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 µmole of denatured Ribonuclease B in one minute at 37˚C, pH 5.5. Cleavage is monitored by SDS-PAGE (cleaved Ribonuclease B migrates faster). SpecificityEndo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.FormulationThe enzyme is provided as a sterile-filtered solution in 20 mM Tris-HCl, 25mM NaCl, 1 mM EDTA (pH 7.5).StabilitySeveral days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. Quality & PurityEndo H is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases.Directions for use1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 37.5 µl final volume with de-ionized water.2. Add 10 µl 5x Reaction Buffer 5.5 and 2.5 µl of Denaturation Solution. Heat at 100°C for 5 minutes.NOTE: It is not necessary to add Triton X-100. SDS will not inactivate Endo H.3. Add 2.0 µl of Endo H to the reaction. Incubate 3 hours at 37°C.If SDS or heat denaturation is omitted, increase incubation time to at least 24 hours.Monitor cleavage by SDS-PAGE.The production host strain has been extensively tested and does not produce any detectable glycosidases... Read More | 6-Bromo-2-naphthyl β-D-glucuronide is a histochemical substrate for β-D-glucuronidase | 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 | Inquire | Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description: High-mobility group box 1 protein (HMGB1), also known as HMG-1 or amphoterin previously, is a member of the HMGB family consisting of three members, HMGB1, HMGB2, and HMGB3. HMGB1 is a DNA-binding nuclear protein,Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description: High-mobility group box 1 protein (HMGB1), also known as HMG-1 or amphoterin previously, is a member of the HMGB family consisting of three members, HMGB1, HMGB2, and HMGB3. HMGB1 is a DNA-binding nuclear protein, released actively following cytokine stimulation as well as passively during cell death. It is the prototypic damage-associated molecular pattern (DAMP) molecule and has been implicated in several inflammatory disorders. HMGB1 signals via the receptor for advanced glycation end-product (RAGE) and members of the toll-like receptor (TLR) family. The most prominent HMGB1 protein and mRNA expression arthritis are present in pannus regions, where synovial tissue invades articular cartilage and bone. HMGB1 promotes the activity of proteolytic enzymes, and osteoclasts need HMGB1 for functional maturation. As a non-histone nuclear protein, HMGB1 has a dual function. Inside the cell, HMGB1 binds DNA, regulating transcription, and determining chromosomal architecture. Outside the cell, HMGB1 can serve as an alarmin to activate the innate system and mediate a wide range of physiological and pathological responses. Extracellular HMGB1 represents an optimal " necrotic marker" selected by the innate immune system to recognize tissue damage and initiate reparative responses. However, extracellular HMGB1 also acts as a potent pro-inflammatory cytokine that contributes to the pathogenesis of diverse inflammatory and infectious disorders. HMGB1 has been successfully therapeutically targeted in multiple preclinical models of infectious and sterile diseases including arthritis. As shown in studies on patients as well as animal models, HMGB1 can play an important role in the pathogenesis of the rheumatic disease, including rheumatoid arthritis, systemic lupus erythematosus, and polymyositis among others. Besides, enhanced postmyocardial infarction remodeling in type 1 diabetes mellitus was partially mediated by HMGB1 activation... Read More |