| Description | EnzymoPure™M-MuLV Reverse Transcriptase (RNase H-) is an optimized Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase without ribonuclease H (RNase H) activity. It is a DNA polymerase that uses single stranded RNA or DNA as template to synthesize complementary DNA strands in the EnzymoPure™M-MuLV Reverse Transcriptase (RNase H-) is an optimized Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase without ribonuclease H (RNase H) activity. It is a DNA polymerase that uses single stranded RNA or DNA as template to synthesize complementary DNA strands in the presence of primers. Different from common M-MuLV reverse transcriptase, the EnzymoPure™M-MuLV Reverse Transcriptase (RNase H-) lacks RNase H activity, and does not degrade the RNA strand of an RNA-DNA hybrid, thus facilitating the synthesis of long cDNA. EnzymoPure™M-MuLV reverse transcriptase (RNase H-) is one of the most widely used reverse transcriptase for synthesizing cDNA.FeaturesApplication：First strand cDNA synthesis using total RNA or mRNA as template; DNA probe labeling; RNA analysis by primer extension; fluorescent probe labeling for DNA microarray analysis.Source：Recombinant protein expressed in E. coli. The RT M-MuLV reverse transcriptase (RNase H-) is encoded by the mutation-optimized pol gene encoding M-MuLV reverse transcriptase.Enzyme activity: One unit of the enzyme incorporates 1nmol of dTMP into a polynucleotide fraction in 10min at 37℃. Enzyme activity is assayed in 50mM Tris-HCl (pH8.3), 6mM MgCl2, 10mM DTT, 40mM KCl, 0.5mM dTTP, 0.4MBq/ml [3H]-dTTP, 0.4mM poly(A)•oligo(dT)12-18.Purity: Free from DNA endonuclease, DNA exonuclease, phosphoesterase and RNase.Storage buffer: 50mM Tris (pH8.3), 100mM NaCl, 1mM EDTA, 5mM DTT, 0.1% Triton X-100 and 50% glycerol.Reaction Buffer (5X): 250mM Tris (pH8.3 at 25℃), 250mM KCl, 20mM MgCl2, 50mM DTT.Inactivation or inhibition：RT M-MuLV Reverse Transcriptase (RNase H-) can be inactivated by incubation at 70℃ for 10 minutes, or inhibited by EDTA, EGTA, inorganic phosphates, pyrophosphates and polyamine.The concentration of this product is 200U/µl. When using a reaction volume of 20µl, this product is sufficient for 10 reactions.Precautions：Please refer to the instructions for reverse transcription of RNAs with high GC content.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear lab coat and disposable gloves during the operation.Instructions for Use：1. First-strand cDNA synthesisa. Set up the first-strand cDNA synthesis reaction in a nuclease-free PCR tube on ice or at room temperature as follows. RNase Inhibitor and dNTP mix can be purchased from. RNA Template (one of the three types of RNA)Total RNA0.01-5µgPoly(A) RNA/mRNA1-500ngSpecific RNA0.01pg-500ngPrimer (one of the three types of primers)Oligo(dT)18 Primer0.5µg (or 100pmol)Random Hexamer Primer0.2µg (or 100pmol)Gene-specific Primer15-25pmol(optional) For RNAs with high GC content or complex secondary structures, incubate the mixture of primer and template at 65ºC for 5 minutes, and immediately put it on ice to disrupt RNA secondary structures.DEPC-treated WaterTo 13.7µl*Reaction Buffer (5X)4µlRNase Inhibitor (40U/µl)0.5µl**dNTP Mix (25 mM each)0.8µl***RT M-MuLV Reverse 1µlTotal Volume20µl* ‘To 13.7µl’ means filling the mixture of template and primer to a total volume of 13.7µl with DEPC-treated water.** The volume of RNase Inhibitor may vary depending on the type of RNase Inhibitor used. If the volume of RNase Inhibitor is less than 0.5µl, adjust the volume of DEPC-treated water accordingly.*** The volume of dNTP mix varies depending on the concentration of dNTP stock. If the volume of dNTP is not 0.8µl, adjust the volume of DEPC-treated water accordingly.b. Mix the reaction by vortex or pipetting gently, centrifuge briefly to allow liquid to accumulate at the bottom of PCR tube.c. Incubate the reaction at 42ºC for 10-60min if Oligo(dT)18 or gene-specific primer is used. If random hexamer is used, carry out incubation at 25ºC for 10min, followed by incubation at 42℃ for 60 min. Note: For RNA template with high GC content or secondary structures, incubate the reaction at 45℃ for 60min.d. Stop the reverse transcription by incubating the reaction at 70℃ for 10min to inactivate the RT M-MuLV Reverse Transcriptase (RNase H-). Note: Heat-inactivation of reverse transcriptase is not recommended for long cDNA over 5kb, as this method may cause shearing of long cDNA fragments. In such a case, phenol-chloroform extraction or column purification can be considered.e. The reverse transcription products can be used directly for subsequent experiments such as PCR, or stored at -20℃ for future use. We recommend using 2µl reverse transcription products in a PCR reaction volume of 50µl.2. For other applications such as primer extension and probe labeling, please refer to reference related to M-MuLV reverse transcriptase (RNase H-)FAQ:1. The reverse transcription product of total RNA is invisible after electrophoresis.It is a normal phenomena, because the amount of RNA template is low, and the amount of reverse transcription products in different size is even lower. 2. No specific product can be amplified from the reverse transcription product.a. To exclude the problem of PCR reaction system or reverse transcription product, use gene-specific primers to amplify internal reference genes, such as actin and GAPDH. Reference genes can be amplified but not the target gene, indicating primers of target gene are not well designed or the expression of the target gene is too low to be detected. b. Inappropriate primer is used for reverse transcription. Random hexamer instead of Oligo(dT)18 should be used for the reverse transcription of bacterial total RNA which does not have poly(A) tails. Gene-specific primers used for reverse transcription must be well designed... Read More | Protein Purity≥85% by SDS PAGEExtinction CoeffA280 nm = 0.631 at 1.0 mg/ml for pure C1qMolecular Weight400,000 Da (18 chains)General DescriptionRat C1q is purified from pooled normal rat serum. C1q is part of the C1 complex, which is the first complement component in the classical pathway of Protein Purity≥85% by SDS PAGEExtinction CoeffA280 nm = 0.631 at 1.0 mg/ml for pure C1qMolecular Weight400,000 Da (18 chains)General DescriptionRat C1q is purified from pooled normal rat serum. C1q is part of the C1 complex, which is the first complement component in the classical pathway of complement. The C1 complex is a non-covalent assembly of three different proteins (C1q, C1r, and C1s) bound together in a calcium-dependent complex. C1q has six extended arms with domains at the end of each arm that bind to the Fc domains of immunoglobulins such as IgG or IgM. When antibodies bind toantigens, forming immune complexes, they cluster allowing two or more of the six C1q arms to bind to the Fc domains of antibodies. Rat IgG2 is very efficient when compared to IgG1 in activating complement (Medgyesi, G.A et., al., 1981). This is in contrast to the human system in which IgG1 activates complement but not IgG2 (Redpath, S. et. al., 1998). The binding of multiple arms of C1q to immune complexes causes the two C1r proteins in the complex (protease zymogens) to auto-activate. The activated C1r proteases cleave and activate the two C1s protease zymogens in the complex. The activated C1s cleaves complement component C4 releasing C4a and initiating covalent attachment of C4b to the activating surface. Activated C1s also cleaves C2 and the larger fragment of C2 binds to the surface-attached C4b forming C4b,C2a, the C3/C5 convertase of the classical pathway.Rat IgG1 cannot activate complement whereas rat IgG2 does.Physical Characteristics & StructureThe apparent molecular weight of rat C1q as determined by gel filtration has been reported to be 400,000 by Veerhuis, R. et al., (1985) and is calculated to be 420,000 based on its amino acid sequence. Rat C1q is a high molecular weight complex of 18 polypeptide chains. Each of the six arms of rat C1q contains three chains, an A chain (~30,000 daltons), a B chain (~28,000 daltons) and a C chain (~26,000 daltons) as determined by SDS/polyacrylamide gel electrophoresis (Wing, M.G. et al., (1993)).FunctionThe biological functions of C1q are described above in the General Description and Physical Characteristics sections.ApplicationsRat C1q can be used to coat ELISA plates to capture and quantitate immune complexes in samples from rat models used for studying immune complex related diseases and conditions.GeneticsNCBI Gene ID numbers for rat C1q are: C1q A chain (298566), C1q B chain (29687), and C1q C chain (362634). The genes for C1q chains A, B and C are all located on chromosome 5. The UniprotKB primary accession numbers for rat C1q are: C1q A chain (P31720), C1q B chain (P31721), and C1q C chain (P31722).Precautions/Toxicity/HazardsThis protein is purified from animal plasma/serum and therefore precautions appropriate for handling any animal blood-derived product must be used.ReferencesMedgyesi, G.A et., Miklos, K., Kulics, J., Fust, G., and Gergely, J. Bazin, H. (1981). Classes and subclasses of rat antibodies: reaction with the antigen and interaction of the complex with the complement system. Immunology 43, 171-176.Redpath, S., Michaelsen, T., Sandlie, I. and Clark, M. R. (1998). Activation of complement by human IgG1 and human IgG3 antibodies against the human leucocyte antigen CD52. Immunology 93, 595–600.Veerhuis, R., Van Es, L.A. and Daha, M.R. (1985). In vivo degradation of rat C1q induced by intravenous injection of soluble IgG aggregates. Immunology 54, 801-810.Wing, M.G., Seilly, D. J., Bridgman, D.J. and Harrison, R.A. (1993). Rapid isolation and biochemical characterization of rat C1 and C1q. Molecular Immunology 30, 433-440... Read More | Carboxypeptidase B catalyzes hydrolysis of the basic amino acids lysine, arginine and histidine from theC-terminal end of polypeptides. The molecular weight is 34,500 daltons, the pH optimum is 8.0, and pI is 6.0.Carboxypeptidase B is competitively inhibited by arginine and lysine. The enzyme is Carboxypeptidase B catalyzes hydrolysis of the basic amino acids lysine, arginine and histidine from theC-terminal end of polypeptides. The molecular weight is 34,500 daltons, the pH optimum is 8.0, and pI is 6.0.Carboxypeptidase B is competitively inhibited by arginine and lysine. The enzyme is also inhibited by metal chelating agents, e.g., EDTA. Recombinant Carboxypeptidase B (EC 3.4.17.2) is expressed in E.Coli and purified by high pressure liquid chromatography. There is no trace of other enzyme (such as carboxypeptidase A and chymotrypsin) activity. No protease inhibitors such as PMSF are present in the preparation.Animal origin free:eliminate the risk of virus presence, or of any other potential adventitious agents found in animal-derived carboxypeptitase B.Stability:A sterile recombinant carboxypeptidase B lyophilized eliminates the risk of contamination and decreases the chances of activity loss in the process of transport and storage. High purity:1) Recombinant carboxypeptidase B provides increased specific activity and eliminates contaminating protease activities found in extracted enzymes with lower purity level. 2) No other contaminating proteases such as chymotrypsin and carboxypeptidase A. 3）Less than 10ppm of recombinant trypsin... Read More | Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：CD200 R1, also known as OX-2 receptor, is a 90 kDa transmembrane protein in the immunoglobulin superfamily and is important in the regulation of myeloid cell activity. The human CD200 R1 cDNA encodes a 325 Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：CD200 R1, also known as OX-2 receptor, is a 90 kDa transmembrane protein in the immunoglobulin superfamily and is important in the regulation of myeloid cell activity. The human CD200 R1 cDNA encodes a 325 amino acid (aa) precursor that includes a 28 aa signal sequence, a 215 aa extracellular domain (ECD), a 21 aa transmembrane segment, and a 61 aa cytoplasmic domain. The ECD is composed of one Ig-like V-type domain and one Ig-like C2-type domain. Within the ECD, human CD200 R1 shares 56% aa sequence identity with both mouse and rat CD200 R1. Alternate splicing of the human CD200 R1 mRNA generates four isoforms, two of which are truncated in the Ig-C2 domain and are likely secreted. In human, a separate CD200 RL gene encodes a protein that shares 81% ECD aa identity with CD200 R1. In mouse, at least four genes for CD200 R1-like molecules have been described. CD200 R1 expression is restricted primarily to mast cells, basophils, macrophages, and dendritic cells, while its ligand, CD200, is widely distributed. Disruption of this receptor-ligand system by knockout of the CD200 gene in mice leads to increased macrophage number and activation and predisposition to autoimmune disorders. Association of CD200 with CD200 R1 takes place between their respective N-terminal Ig-like domains. The capacity of CD200 R1-like molecules to interact with CD200 is controversial. CD200 R1 propagates inhibitory signals despite lacking a cytoplasmic ITIM (immunoreceptor tyrosine-based inhibitory motif). CD200 R1-like molecules, in contrast, are potentially activating receptors by means of their association with DAP12. CD200R1 signaling inhibits the expression of proinflammatory molecules including TNFs, IFNs, and inducible nitric oxide synthase in response to selected stimuli, which implicate that CD200/CD200R1 inhibitory signaling pathway plays a prominent role in limiting inflammation in a wide range of inflammatory diseases. Furthermore, the CD200/CD200R inhibitory signaling constitutes one of the most suitable endogenous immunoregulatory molecule candidate to restore the immune suppressive status of the CNS altered in chronic neuroinflammatory situations... Read More | Purity≥95% SDS-PAGE. Recombinant human MIF, fused to His-tag at N-terminus, was cloned into an E. coli expression vector and was purified to apparent homogeneity by using conventional column chromatography techniques.FunctionPro-inflammatory cytokine. Involved in the innate immune response to Purity≥95% SDS-PAGE. Recombinant human MIF, fused to His-tag at N-terminus, was cloned into an E. coli expression vector and was purified to apparent homogeneity by using conventional column chromatography techniques.FunctionPro-inflammatory cytokine. Involved in the innate immune response to bacterial pathogens. The expression of MIF at sites of inflammation suggests a role as mediator in regulating the function of macrophages in host defense. Counteracts the anti-inflammatory activity of glucocorticoids. Has phenylpyruvate tautomerase and dopachrome tautomerase activity (in vitro), but the physiological substrate is not known. It is not clear whether the tautomerase activity has any physiological relevance, and whether it is important for cytokine activity... Read More |