| Description | EnzymoPure™Q M-MLV Reverse Transcriptase uses single-stranded RNA or DNA in the presence of a primer to synthesize a complementary DNA strand with high fidelity, sensitivity, and specificity. It is a modified, optimized reverse transcriptase particularly suitable for qPCR analysis. The EnzymoPure™Q M-MLV Reverse Transcriptase uses single-stranded RNA or DNA in the presence of a primer to synthesize a complementary DNA strand with high fidelity, sensitivity, and specificity. It is a modified, optimized reverse transcriptase particularly suitable for qPCR analysis. The EnzymoPure™Q M-MLV Reverse Transcriptase contains RNase H enzyme activity that can specifically degrade the RNA strand of an RNA-DNA hybrid, enabling the subsequent synthesis of second-strand cDNA.EnzymoPure™Q M-MLV Reverse Transcriptase, a commonly used high-quality reverse transcriptase, is widely used in the synthesis of first-strand cDNA using total RNA or mRNA templates, and is particularly suitable for qPCR and one-step qRT-PCR analysis. This product is also applicable for conventional PCR, synthesis of second-strand cDNA and construction of cDNA library, and cloning of target genes. It can also be used for DNA probe labeling with fluorescence, biotin, digoxin, or isotope through reverse transcription reaction, or for RNA studies by primer extension.Please refer to Figure 1 for the qPCR analysis of the GADPH gene in HEK293T cells using the cDNA templates obtained by the EnzymoPure™Q M-MLV reverse transcriptase.Figure 1Source：Recombinant reverse transcriptase expressed in E. coli.Definition of enzyme activity unit: One unit is defined as the amount of enzyme that incorporates 1 nmol of dTTP into acid-precipitable material in 10min at 37℃ using poly(A)•oligo(dT)12-18 as template-primer. Reaction system: 50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl2, 10mM DTT, 0.5mM [3H]-dTTP, and 0.4mM polyA•oligo(dT)12-18.Purity: This product is free from DNA endonuclease, exonuclease, phosphatase, and RNase, and can meet the requirements of conventional reverse transcription.Storage buffer: 20mM Tris-HCl (pH7.5), 150mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01%(v/v) NP-40, and 50%(v/v) glycerolInactivation or inhibition：RT Q M-MLV Reverse Transcriptase can be inactivated by incubation at 80℃ for 10 minutes, or inhibited by EDTA and EGTA chelators, inorganic phosphates or pyrophosphates, and polyamine.The concentration of M-MLV is 200U/µl. When 20µl of reverse transcription reaction volume is used, D7188S, D7188M, and D7188L are sufficient for 50, 250, and 1000 reactions, respectively. provides a variety of reverse transcriptase. To select the best one for your needs, please refer to the following webpage:http://www.aladdin-e.com/support/reversetranscriptase.htmPrecautions：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 a lab coat and disposable gloves during the operation.Instructions for Use：1. First-strand cDNA synthesisa. Set up the following reaction on ice or at room temperature. RNase Inhibitor and dNTP mix can be purchased from SYBR Green qPCR Mix (2X), SYBR Green qPCR Mix (2X, Low ROX), and SYBR Green qPCR Mix (2X, High ROX) are for conventional qPCR analysis with SYBR Green fluorescent dye. Probe qPCR Mix (2X), Probe qPCR Mix (2X, Low ROX), and Probe qPCR Mix (2X, High ROX) are for qPCR analysis with Taqman Probe.b. Perform qPCR analysis following the instructions of the corresponding qPCR kit.3. For other applications such as primer extension and probe labeling, please refer to references related to M-MLV reverse transcriptase.FAQ:1. The reverse transcription product of total RNA is invisible after electrophoresis.It is a normal phenomenon, because the amount of RNA template is low, and the amount of reverse transcription products in different sizes is even lower. 2. No specific product is 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. If reference genes can be amplified, but not the target gene, it indicates primers of the target gene are not well designed. b. Template RNA may be degraded. The integrity of total RNA can be checked by agarose gel or on-chip electrophoresis. Intact total RNA exhibits sharp, clear 28S and 18S rRNA bands, and the 28S rRNA band should be approximately twice as intense as the 18S rRNA band. A ratio less than 2 indicates the degradation of total RNA and new total RNA should be prepared. c. RNA samples may contain some components that inhibit the activity of reverse transcriptase. Those contaminants include phenol, SDS, EDTA, guanidine salts, phosphoric acid, pyrophosphoric acid, polyamine, spermidine, etc, which can be removed effectively by column purification or precipitation, washing, and redissolution. Total RNA extracted by Zol or Trizol produced by can fully meet the requirements of reverse transcription.d. Insufficient templates for reverse transcription. When DNase I is used to remove the residual DNA in the RNA sample prior to reverse transcription and is subjected to heat-inactivation, EDTA should be added to the RNA sample at a final concentration of 2.5mM to protect RNA from degradation under high temperature. Additionally, to amplify a specific gene, it is necessary to extract RNA from tissues in which the target gene is highly expressed. e. An 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 | Amino Acid Sequence Asn-Ser-Lys-Met-Ala-His-S?er-Ser-Ser-Cys-Phe-Gly-Gl?n-Lys-Ile-Asp-Arg-Ile-Gly?-Ala-Val-Ser-Arg-Leu-Gly-?Cys-Asp-Gly-Leu-Arg-Leu-P?he | 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 | 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 | Human PTHrP-(1-36) is a secretory form of PTHrP with anticalciuric effects. Human PTHrP-(1-36) enhances beta cell function and proliferation. Human PTHrP-(1-36) can be used in the research of humoral hypercalcemia of malignancy (HHM) and hyperparathyroidism.In VitroHuman PTHrP-(1-36) (EC 50 : 0.05 Human PTHrP-(1-36) is a secretory form of PTHrP with anticalciuric effects. Human PTHrP-(1-36) enhances beta cell function and proliferation. Human PTHrP-(1-36) can be used in the research of humoral hypercalcemia of malignancy (HHM) and hyperparathyroidism.In VitroHuman PTHrP-(1-36) (EC 50 : 0.05 nM) increases intracellular calcium in human epidermal keratinocytes. Human PTHrP-(1-36) (100 nM, 24 h) increases human β-cell proliferation. Human PTHrP-(1-36) (100 nM, 30 min) enhances insulin secretion in human islets. PTHrP-(1-36) (mouse, EC 50 : 1 nM) induces a rapid Ca 2+ response in UMR 106 cells. MCE has not independently confirmed the accuracy of these methods. They are for reference only.In VivoPTHrP-(1-36) (mouse, 160 µg/kg, s.c., for 5 days/week for 7, 30, or 90 days) enhances beta cell regeneration and increases beta cell mass in a mouse model of partial pancreatectomy. PTHrP-(1-36) (mouse, 100 µg/kg, s.c., every other day) reverses the observed decrease of Wisp1 expression in the diabetic mice. MCE has not independently confirmed the accuracy of these methods. They are for reference only.Form:Solid... Read More |