| Description | EnzymoPure™II M-MLV Reverse Transcriptase (RNase H-) produced by aladdin is an optimized Moloney Murine Leukemia Virus (M-MLV) 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 EnzymoPure™II M-MLV Reverse Transcriptase (RNase H-) produced by aladdin is an optimized Moloney Murine Leukemia Virus (M-MLV) 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. It is one of the most widely used reverse transcriptase.sApplication：First strand cDNA synthesis using total RNA or mRNA as template; DNA probe labeling with fluorescence, biotin, digoxin or isotope; RNA analysis by primer extension.This product is highly cost-effective. The RT II M-MLV reverse transcriptase (RNase H-) is a recombinant reverse transcriptase expressed and purified from E. coli transformed with the expression plasmids carrying the pol gene fragment of M-MLV with RNase H coding region removed. The enzyme has been engineered to have better thermal stability and higher reverse transcription activity.Definition of enzyme activity: One unit of the enzyme incorporates 1nmol of dTTP into acid-precipitable material in 10 min at 37℃ using poly(A)•oligo(dT)12-18 as template-primer. The reaction system contains 50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl2, 10mM DTT, 0.5mM [3H]-dTTP and 0.4 mM poly(A)•oligo(dT)12-18.Purity: Free from DNA endonuclease, DNA exonuclease, phosphoesterase and RNase.Enzyme storage buffer: 20mM Tris-HCl (pH7.5), 300mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01%(v/v) NP-40 and 50% (v/v) glycerol.Inactivation or inhibition: RT II M-MLV Reverse Transcriptase (RNase H-) can be inactivated by incubation at 80ºC for 10 minutes, or inhibited by EDTA, EGTA, inorganic phosphates, pyrophosphates and polyamine.This product has high reverse transcription activity, and is able to synthesize long cDNA. The RT II M-MLV 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. It can easily synthesize cDNA up to 8kb (Figure 1). The maximum length of synthesized cDNA by this product can exceed 10kb. Figure 1. Agarose gel electrophoresis of PCR products amplified from the cDNA template that was synthesized by the RT II M-MLV reverse transcriptase (RNase H-) (, #). cDNA ranging from 0.2kb to 8kb in length can be synthesized high efficiently.This product exhibits high thermal stability. This product has an optimum working temperature of 42-45ºC, but it is still highly active at 50ºC (Figure 2). Figure 2. Agarose gel electrophoresis of PCR products amplified from the cDNA templates that were synthesized by the RT II M-MLV reverse transcriptase (RNase H-) (, #) and by the M-MLV reverse transcriptase (RNase H-) (Beytome, ), respectively. 500ng of total RNA extracted from NIH3T3 cells was reverse transcribed by reverse transcriptase in a reaction volume of 20µl at difference temperatures as indicated in the figure. One microliter of each reverse transcription reaction product was taken for PCR amplification.The concentration of this product is 200U/µl. When 20µl of reverse transcription reaction volume is used, the S, M and L are sufficient for 50, 250 and 1000 reactions, respectively.Enzyme storage buffer：20mM Tris-HCl (pH7.5), 300mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01%(v/v) NP-40 and 50% (v/v) glycerol.Inactivation or inhibition：RT II M-MLV Reverse Transcriptase (RNase H-) can be inactivated by incubation at 80ºC for 10 minutes, or inhibited by EDTA, EGTA, inorganic phosphates, pyrophosphates and polyamine.This product has high reverse transcription activity, and is able to synthesize long cDNA. The RT II M-MLV 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. It can easily synthesize cDNA up to 8kb (Figure 1). The maximum length of synthesized cDNA by this product can exceed 10kb. Figure 1. Agarose gel electrophoresis of PCR products amplified from the cDNA template that was synthesized by the RT II M-MLV reverse transcriptase (RNase H-). cDNA ranging from 0.2kb to 8kb in length can be synthesized high efficiently.This product exhibits high thermal stability. This product has an optimum working temperature of 42-45ºC, but it is still highly active at 50ºC (Figure 2). Figure 2. Agarose gel electrophoresis of PCR products amplified from the cDNA templates that were synthesized by the RT II M-MLV reverse transcriptase (RNase H-) and by the M-MLV reverse transcriptase (RNase H-) (Beytome, D7159), respectively. 500ng of total RNA extracted from NIH3T3 cells was reverse transcribed by reverse transcriptase in a reaction volume of 20µl at difference temperatures as indicated in the figure. One microliter of each reverse transcription reaction product was taken for PCR amplification.The concentration of this product is 200U/µl. When 20µl of reverse transcription reaction volume is used, the D7160S, D7160M and D7160L are sufficient for 50, 250 and 1000 reactions, respectively.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 synthesis.a. 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. Template (one of the three types of RNA)Total RNA0.1ng-5µgPoly(A) RNA/mRNA10pg-0.5µgSpecific RNA0.01pg-0.5µgPrimer (one of the three types of primer)Oligo(dT)18 Primer0.5µg (or 100pmol)Random Hexamer Primer0.2µg (or 100pmol)Gene-specific Primer15-25pmol(Optional) For RNAs with high GC content (e.g. ＞55%) 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 Water-To 13.7µl*Reaction Buffer (5X)-4µlRNase Inhibitor (40U/µl)-0.5µldNTP Mix (25mM each)-0.8µl**RT II M-MLV Reverse Transcriptase (RNase H-)-1µl***Total Volume-20µl* ‘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 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.*** If gene specific primers or Oligo(dT)18 primers are used to reverse transcribe the cDNA over 5kb, the volume of RT II M-MLV reverse transcriptase (RNase H-) should be increased to 2µl.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 60min if Oligo(dT)18 primer or gene-specific primer is used. If random hexamer is used, carry out incubation at 25℃ for 10min, followed by incubation at 42℃ for 60min. Note: For RNA template with high GC content or secondary structures, incubate the reaction at 50℃ for 60min in order to improve the reverse transcription efficiency.d. Stop the reverse transcription by incubating the reaction at 80ºC for 10min to inactivate RT II M-MLV 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. If reference genes can be amplified but not the target gene, it indicates primers of target gene are not well designed, the expression of the target gene is too low to be detected, or the reverse transcription efficiency is low. 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 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, and spermidine etc, which can be removed effectively by column purification or precipitation, washing, and redissolution. Total RNA extracted by Zol (, #R0011) or Trizol (, #R0016) usually can meet the requirements of reverse transcription.d. Insufficient templates for reverse transcription. When DNase I used to remove the residual DNA in RNA sample is subjected to heat-inactivation prior to reverse transcription, EDTA should be added to 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. 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.f. For RNA template with high GC content or secondary structures, increase the reverse transcription temperature to 45-50℃ to improve the reverse transcription efficiency... Read More | 6-Bromo-2-naphthyl β-D-glucuronide is a histochemical substrate for β-D-glucuronidase | 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 | Product Characteristics UNI-StabilPLUS is a universal stabilizer for the dilution and stabilization of both Horseradish Peroxidase (HRP) and Alkaline Phosphatase (AP) labeled proteins and antibodies, in order to maintain the molecular conformation and prevent loss of activity over time. This enablesProduct Characteristics UNI-StabilPLUS is a universal stabilizer for the dilution and stabilization of both Horseradish Peroxidase (HRP) and Alkaline Phosphatase (AP) labeled proteins and antibodies, in order to maintain the molecular conformation and prevent loss of activity over time. This enables the making of pre-diluted, ready-to-use conjugates, minimizing assay errors in dilution. Superior stabilization of HRP and AP conjugated antibodies in low as well as high protein dilutions is seen, when using UNI-StabilPLUS. When tested with AP conjugated antibody stability is seen as follows: • at least 3 years at 2-8 °C • at least 2 years at room temperature • at least 4 weeks at 37 °C When tested with HRP conjugated antibody stability is seen as follows: • at least 2 years at 2-8 °C • at least 1 years at room temperature • at least 2 weeks at 37 °CUNI-StabilPLUS is recommended for the dilution of antibodies directed against rabbit immunoglobulins unlike HRP-StabilPLUS (cat. no. H494387) and Antibody Enhancer (cat. no. A494276).Composition & Properties UNI-StabilPLUS is a ready-to use buffer that appears as an opaque solution. The product is based on a mild acid Tris buffer containing proprietary stabilizing components. UNI-StabilPLUS contains neither BSA, nor other material from bovine serum, no azide, mercury or other toxic components.Working Procedure 1.Make a series of dilutions of the HRP- or AP conjugated protein in UNI-StabilPLUS in order to determine the optimal dilution. 2.Run the assay as usual or store the diluted conjugated protein preferably at 2-8 °C.Tips & Tricks • Avoid using phosphate buffers for AP-conjugated antibody assays. We recommend the use of Tris/HCl, Tween as the washing buffer, instead of a PBS buffer which will reduce signal significantly. • For extended stability of HRP conjugated antibodies, HRP-StabilPLUS (cat. no. H494387) is recommended. Handling & Storage • Store solution at 2-8 °C... Read More |