| Description | One unit is defined as the amount of enzyme required to ligate 50% of HindIII fragments of λ DNA (5' DNA termini concentration of 0.12µM, 300µg/ml) in a total reaction volume of 20µl in 30 minutes at 16℃.Application：Ligation of dsDNA with sticky ends; nick repair of One unit is defined as the amount of enzyme required to ligate 50% of HindIII fragments of λ DNA (5' DNA termini concentration of 0.12µM, 300µg/ml) in a total reaction volume of 20µl in 30 minutes at 16℃.Application：Ligation of dsDNA with sticky ends; nick repair of dsDNA; cloning after the synthesis of second strand cDNA [2]. Source：Recombinant E. coli DNA ligase expressed in E. coli.Enzyme storage buffer：10mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 200µg/ml BSA, 50% Glycerol (pH7.4, 25℃).Inactivation or inhibition：E. coli DNA Ligase can be inactivated by incubation at 65℃ for 20 minutes.Precautions：The ligation efficiency of this product for blunt-end fragments is extremely low. Use T4 DNA Ligase for blunt-end ligation.This product cannot be used for the ligation of ssDNA or RNA.This kit is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1. Set up the reaction on ice as follows:ReagentVolumeFinal ConcentrationdsDNAxµlup to 0.25µg/µl10X Reaction Buffer2µl1XE. coli DNA Ligase (10U/µl)1µl0.5U/µlNuclease-free Water(17-x)µl-Total Volume20µl-Note 1: When multiple reactions are required, prepare a master mix including all reagents except the substrate DNA and then dispense to different nuclease-free PCR tubes. Finally, add substrate DNA to each tube.Note 2: E. coli DNA Ligase should be kept on ice during the experiment.2. Mix well by pipetting or vortex gently. Centrifuge briefly to collect liquid at the bottom of the PCR tube.3. Incubate at 16℃ for 30-60 minutes.4. After incubation, immediately heat inactivate the reaction at 65℃ for 20 minutes.5. Examine the reaction products by agarose gel or polypropylene gel electrophoresis. If DNA need to be recovered from agarose gel, we recommend using the DNA Gel Extraction Kit. To purify DNA from the enzyme digestion reactions, we recommend using the PCR Clean Up Kit/DNA Purification Kit. 6. For other applications, please refer to the relevant literature for E. coli DNA Ligase.FAQ:1. What is the difference between E. coli DNA Ligase and T4 DNA Ligase?Under the recommended conditions in the manual, E. coli DNA Ligase cannot ligate blunt-end DNA or RNA molecules [3].2. Can E. coli DNA Ligase be heat-inactivated?E. coli DNA Ligase can be inactivated by incubation at 65℃ for 20 minutes.3. How to choose the appropriate ligase or ligation kit?For rapid ligation of blunt- or sticky-end DNA fragments, the Rapid DNA Ligation Kit is recommended. T4 DNA Ligase is suitable for most DNA recombination reactions and can be used for sticky-end (10-minute ligation at room temperature) or blunt-end (2-hour or overnight ligation at room temperature) ligations. However, E. coli DNA Ligase is more selective for substrates and is recommended for sticky-end ligations. For ligation of ssDNA or RNA molecules, we recommend using the T4 RNA Ligase. References:1. Lehman IR. Science. 1974. 186(4166):790-7.2. Okayama H and Berg P. Mol Cell Biol. 1982. 2(2):161-70.3. Higgins NP and Cozzarelli NR. Methods Enzymol. 1979. 68:50-71... 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 | Inquire | Inquire | Purity> 96% by SDS-PAGE and HPLC analyses.FunctionHas weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca(2+) changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T-lymphocytes, Purity> 96% by SDS-PAGE and HPLC analyses.FunctionHas weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca(2+) changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T-lymphocytes, neutrophils, and eosinophil leukocytes. Enhances the proliferation of CD34 myeloid progenitor cells. The processed form HCC-1(9-74) is a chemotactic factor that attracts monocytes eosinophils, and T-cells and is a ligand for CCR1, CCR3 and CCR5.Post-translationalThe N-terminal processed forms HCC-1(3-74), HCC-1(4-74) and HCC-1(9-74) are produced in small amounts by proteolytic cleavage after secretion in blood. HCC-1(1-74), but not HCC-1(3-74) and HCC-1(4-74), is partially O-glycosylated; the O-linked glycan consists of one Gal-GalNAc disaccharide, further modified by two N-acetylneuraminic acids... Read More |