| Description | CANT1 Human Pre-designed siRNA Set A contains three designed siRNAs for CANT1 gene (Human), as well as a negative control, a positive control, and a FAM-labeled negative control. Components CANT1 siRNA-1: 5 nmol (HPLC) CANT1 siRNA-2: 5 nmol (HPLC) CANT1 siRNA-3: 5 nmol (HPLC) siRNA Negative Control:CANT1 Human Pre-designed siRNA Set A contains three designed siRNAs for CANT1 gene (Human), as well as a negative control, a positive control, and a FAM-labeled negative control. Components CANT1 siRNA-1: 5 nmol (HPLC) CANT1 siRNA-2: 5 nmol (HPLC) CANT1 siRNA-3: 5 nmol (HPLC) siRNA Negative Control: 5 nmol (HPLC) FAM-labeled siRNA Negative Control: 5 nmol (HPLC) GAPDH siRNA Positive Control:5 nmol (HPLC)... Read More | Protein Purity>90 % by SDS PAGEExtinction CoeffA280 nm = 0.725 at 1.0 mg/mL for pure C1s-C1INH ComplexMolecular Weight196,000 Da (1 chain)General DescriptionThe product C1s-C1INH Complex is made by interacting purified protease inhibitor C1-INH with purified C1s enzyme followed by purification. Protein Purity>90 % by SDS PAGEExtinction CoeffA280 nm = 0.725 at 1.0 mg/mL for pure C1s-C1INH ComplexMolecular Weight196,000 Da (1 chain)General DescriptionThe product C1s-C1INH Complex is made by interacting purified protease inhibitor C1-INH with purified C1s enzyme followed by purification. The protease inhibitor C1-INH prevents the spontaneous activation of complement and limits consumption of C2 and C4 by rapidly inactivating C1r, C1s and MASP2. It is the only plasma serine protease inhibitor (Serpin) capable of interacting with and inhibiting activated C1. C1-INH interacts with the catalytic sites of both C1r and C1s. The interaction with activated C1r and C1s is covalent resulting in complexes which are stable to SDS. C1s and C1r enzymes, however, are irreversibly inactivated by binding to C1-INH. C1s-C1INH is a very stable complex that remains intact even when subjected to freeze/thaw cycles with almost no loss of the complex form.Physical Characteristics & StructureThe C1s enzyme-C1INH complex is composed of two disulfide linked chains from C1s enzyme (A chain 58,000 Da and B chain 28,000 Da) and one covalently linked chain from C1-INH (75,000 Da).SDS-PAGE analysis of the C1s-C1INH complex shows a single band of about 161,000 Da under nonreducing conditions. Under reducing conditions, the C1s-C1INH complex exhibits two bands: A 58,000 Da band corresponding to the A chain of C1s enzyme and a second 103,000 Da band resulting from C1INH (75,000 Da) covalently bond to the B chain (28,000 Da) of C1s enzyme.RegulationActivated C1s is controlled by C1-INH. C1s enzyme and C1-INH form a covalent complex that is resistant to separation on SDS gels. During complement activation C1 complex is rapidly activated by binding to immune complexes. The resulting activated C1s and C1r are rapidly inactivated by interaction with C1-INH (Ziccardi, R.J. (1982)). Binding to immune complexes is fast (10-20 sec) and activation of the bound C1 complex takes several minutes, but C1-INH has also been shown to be fast and no active C1r or C1s remain 4 min after addition of immune complexes to plasma (Ross, G.D. (1986); Ziccardi,R.J. (1981)). The binding of C1-INH to activated C1 releases both C1r and C1s from the complex leaving C1q bound to the immune complex. The released complexes contain four molecules: C1-INH-C1r-C1s-C1-INH. The reaction of C1 esterase inhibitor with activated C1 is very fast with the estimated half-life of C1r and C1s being approximately 15 seconds in serum. In fact, at serum concentrations of C1- INH little or no additional C4 or C2 activation occurs 3 min after immune complexes are added because all the C1r and C1s molecules have been inactivated and removed from the C1q which remains bound to the immune complex (Ross, G.D. (1986); Morley, B.J. and Walport, M.J. (2000); Rother, K., et al. (1998); Ziccardi, R.J. (1982a and 1982b); Morgan, B.P. (1990)). The interaction of purified C1s enzyme and C1-INH is slower.FunctionSee General Description and Regulation above.ApplicationsC1s-C1INH complex can be used in studies designed for developing and identifying inhibitors of C1s-C1INH complex formation and thus lead to the possible development of therapeutics for inhibiting complement activation via the classical pathway.GeneticsThe EMBL/Genbank cDNA accession number for C1s is J04080. The gene for C1s is located on chromosome 12p13. The EMBL/Genbank cDNA accession numbers for C1-INH are M13656 and X54486 (human) and Y10386 (mouse). The gene for C1-INH is located on chromosome 11p11.2-13. DeficienciesC1s deficient patients are prone to systemic lupus erythematosus (SLE) and recurrent pyogenic infections (Rother, K., et al. (1998)). They lack classical pathway function. The genetic disorder hereditary angioedema (HAE) is caused by a partial deficiency of C1-INH. Patients with HAE have low functional C1-INH levels in blood and have recurrent episodes of systemic or localized edema.DiseasesSee section titled 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.ReferencesZiccardi, RJ. (1982) A new role for C-1-inhibitor in homeostasis: control of activation of the first component of human complement. J. Immunol. 128:2505-2508.Ross, G.D. (1986) Immunobiology of the Complement System. (ISBN 0-12-5976402) Academic Press, Orlando.Ziccardi, R.J. (1981) Activation of the early components of the classical complement pathway under physiologic conditions. J. Immunol. 126:1769-1773.Morley, B.J. and Walport, M.J. (2000) The Complement Facts Book. (ISBN 0127333606) Academic Press, London.Rother, K., Till, G.O., and Hӓnsch, G.M. (1998) The Complement System. (ISBN 3-540- 61894-5) Springer-Verlag, Heidelberg.Ziccardi, R.J. (1982a) Spontaneous activation of the first component of human complement (C1) by an intramolecular autocatalytic mechanism. J. Immunol. 128:2500- 2504.Ziccardi, RJ. (1982b) A new role for C-1-inhibitor in homeostasis: control of activation of the first component of human complement. J. Immunol. 128:2505-2508. Morgan, B.P. (1990) Complement Clinical Aspects and Relevance to Disease. (ISBN 0- 12-506955-3) Academic Press, London... Read More | Protein Purity>95% by SDS-PAGEExtinction Coeff.A276 nm = 0.456 at 1.0 mg/mLMolecular Weight8,759 Da (single chain)General DescriptionNatural human C4a is prepared by cleavage of human C4 protein by human C1s. It is produced during activation of both the classical and lectin pathways of complementProtein Purity>95% by SDS-PAGEExtinction Coeff.A276 nm = 0.456 at 1.0 mg/mLMolecular Weight8,759 Da (single chain)General DescriptionNatural human C4a is prepared by cleavage of human C4 protein by human C1s. It is produced during activation of both the classical and lectin pathways of complement. C4a is a member of the anaphylatoxin family of three proteins (C3a, C4a and C5a) produced by the activation of complement (Hugli, T.E. et al. (1981)). It is an unglycosylated polypeptidecontaining 77 amino acids with a molecular mass of 8,759 daltons. Many of the biological functions of C4a are similar to those of C3a, but the specific activities are far below those of C3a. C4a activity is so low, in fact, that it was initially thought to be inactive. These measured activities include inducing muscle contraction in the guinea pig ileum test (spasmogenic activity), desensitization of muscle to C3a stimulation suggesting that the same receptor for both C3a and C4a is involved (tachyphylactic activity) and inducing vascular permeability in human skin (Gorski J.P. et al. (1979)). C4a does not show tachyphylactic activity against C5a or chemotactic activity. Removal of the C-terminal arginine by serum carboxypeptidase N destroys all these activities (Meuller-Ortiz, S.L., et al. (2009)). C4a appears to act through the C3a receptor (C3aR) which is a G-protein coupled receptor found widely distributed on peripheral tissues, lymphoid cells (neutrohphils, monocyes, and eosinophils) and in the central nervous system (astrocytes, neurons and glial cells) (Law, S.K.A. and Reid, K.B.M. (1995)). Physical Characteristics & StructureMolecular weight: 8,759 calculated molecular mass. Observed mass (MALDI-TOF) is 8,762 + 9 mass units. pI = 9.0 to 9.5 (Gorski, J.P. et al. (1981))Amino acid sequence (77 amino acids): NVNFQKAINE KLGQYASPTA KRCCQDGVTR LPMMRSCEQR AARVQQPDCR EPFLSCCQFA ESLRKKSRDK GQAGLQRC4a is thought to be structurally very similar to C3a and C5a to which it is homologous. Thus its 3D structure is probably similar to the X-ray-derived crystal structureof C3a (Huber, R. et al. (1980)) and the NMR derived structure of C3a: Nettesheim, D.G. et al. (1988); Murray, I. et al. (1999).FunctionSee General Description above. C4a exhibits much weaker biological activities than C3a and C5a. Its activity in inducing erythema and edema in human skin is 25,000-fold weaker than that of C5a and 100-fold weaker than C3a per nanomole. The spasmogenic activity of C4a is 2000-fold weaker than C5a and 100-fold weaker than that of C3a. Due to these differences the role of C4a in these responses in vivo is thought to be negligible.AssaysTwo well established assays for C4a and C3a functional activities include induction of contraction in the guinea pig ileum and the permeation of a dye such as trypan blue from the vasculature into skin. The anaphylatoxins also induce mast cell degranulation, (measured as histamine release), platelet aggregation, IL-1 release from monocytes and the release of prostaglandins and leukotrienes from many cells and tissues. The other assays used for C3a (Dodds, A.W. and Sim, R.B. (1997)) should also respond to C4a, but few reports have described utilizing these assays with C4a. ELISA kits for the assay of C4a levels (or more correctly C4a desArg levels) in blood and other fluids are sold by several 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 vivoFreshly drawn normal human serum contains significant levels of all three anaphylatoxins. Although these may represent the resting concentration in vivo it is difficult to draw or store blood without some complement activation so a true in vivo concentration is difficult to determine. The presence of EDTA and Futhan in the collection tubes can minimize this background (Pfeifer, P.H. et al. (1999)). Full activation of all C4 in blood (600µg/mL) would result in ~3,400 nM C4a (~30 µg/mL). Due to the low biological activity of C4a it could require activation of most of the C4 in a small region to achieve the micromolar C4a concentrations necessary to elicit a response.RegulationC4a levels are regulated by three processes: formation, inactivation and clearance. There are two enzymes that cleave C4 and release C4a: C1s and MASP-2. C4a is “inactivated” by removal of its C-terminal arginine amino acid. The product C4a desArg (or C4a without the C-terminal arginine) is produced by the action of the plasma enzyme carboxypeptidase N (Mueller-Ortiz S.L. et al. (2009)). The inactivation is rapid and most C4a is converted to C4a desArg within minutes of its formation. Inactivated C4a lack measurable biological activity. Because of the large number of cells bearing C3a/C4areceptors (endothelial, immune, smooth muscle, neuronal, etc.) the capture, internalization and digestion of C4a and C4a desArg probably results in its removal from circulation.DeficienciesA deficiency of C4 or a deficiency of all of the enzymes that cleave C4 to generate C4a could result in the absence of C4a. There are no known complete deficiencies of all ofthe C4 cleaving enzymes. Examples of C4 deficient humans and mice exist (Wessels, M.R. et al. (1995)), but the degree to which pathologies associated with C4 deficiency are due to the lack of C4 or the absence of C4a is unclear. DiseasesThere are no known diseases connected to C4a or C4a desArg. Precautions/Toxicity/HazardsThe source of C4a is human serum, therefore appropriate precautions must be observed 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 3... Read More | TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site.The substrate does not contain NMP (1-methyl2-pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal negative environmental impact.Product Characteristics TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site. The substrate does not contain NMP (1-methyl-2- pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal waste problems after use.Composition & Properties Ready-to-use substrate: Includes substrate buffer and hydrogen peroxide. No other reagents should be added.Working Procedure The following procedure is applicable to nitrocellulose membranes. The procedure must be optimized for other membranes.1.The desired amount of substrate is poured into a sealed container and allowed to reach room temperature, in the dark, before use. 2.After the last incubation with HRP-labelled Streptavidin or HRP-labelled secondary antibody it is recommended to wash the membrane in a 0.1 M Tris buffer pH 7.4.3.Shake off the excess buffer and incubate the membrane in the TMB-D Blotting solution for 10 minutes. 4.Wash the membrane in distilled water and allow it to dry. 5.The site of positive reaction will appear light blue with no or very little background staining.Tips & Tricks • The membrane can be blocked with Kementec’s Synthetic Blocking Buffer for Blotting, (cat. no. S494457). • For long-term preservation of the results, the membranes must be stored in the dark.Handling & Storage • Store solution at 2-8⁰C in the dark. • Avoid exposure to light, heat and contamination with metal ions or peroxidase. • Re-dispense only into bottles made of High-Density Polyethylene (HDPE), amber color. Dispensing guidelines are available upon request... Read More | Tyrosine decarboxylase catalyzes the removal of the carboxyl group from tyrosine to produce tyramine and carbon dioxide. Pyridoxal 5'-phosphate is a necessary cofactor. By using the apoenzyme prepared from cells grown on a vitamin B6 deficient medium pyridoxal phosphate may be determined. The Tyrosine decarboxylase catalyzes the removal of the carboxyl group from tyrosine to produce tyramine and carbon dioxide. Pyridoxal 5'-phosphate is a necessary cofactor. By using the apoenzyme prepared from cells grown on a vitamin B6 deficient medium pyridoxal phosphate may be determined. The HOLOenzyme may be used to determine tyrosine, phenylalanine and dihydroxyphenylalanine either manometrically or colorimetrically.L-Tyrosine decarboxylase apoenzyme from Streptococcus faecalis has been used in a study to purify and characterize tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase.L-Tyrosine decarboxylase apoenzyme from Streptococcus faecalis has also been used in a study to investigate the stereospecificity of sodium borohydride reduction of tyrosine decarboxylase.One Unit yields 1µmole of CO2 per minute from L-tyrosine at 37°C, pH 5.5. The APOenzyme activity is measured in the presence of excess pyridoxal phosphate... Read More |