| Description | Product introduction：Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. FirstProduct introduction：Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. First, luciferin was used as substrate to detect the activity of firefly luciferase, then substances inhibiting the catalysis of firefly luciferase were added, and coelenterazine was added to detect the activity of Renilla luciferase to achieve dual luciferase reporter gene detection. The bioluminescence system of luciferase and its substrate can detect gene expression very sensitively and efficiently. Usually, the transcriptional regulatory element or 5'promoter region of the gene of interest is cloned upstream of luciferase, or the 3'-utr region is cloned downstream of luciferase to construct a reporter gene plasmid, and then transfect the cells. After the cells are treated with appropriate drugs, the cells are lysed, and the transcriptional regulation effect of drug treatment on the target gene is judged by detecting the luciferase activity. Renilla luciferase is more often used as an internal reference for detecting transfection efficiency to eliminate the difference in cell number and transfection efficiency. Firefly luciferase is a protein with a molecular weight of about 61 kDa. In the presence of ATP, magnesium ions and oxygen, it can catalyze the production of oxyluciferin from luciferin. In the process of luciferin oxidation, it will produce a light signal. Renilla luciferase is a protein with a molecular weight of about 36 kDa. In the presence of oxygen, it can catalyze the oxidation of coelenteramide to coelenteramide, and also produce light signals in the process of coelenteramide oxidation. The optical signal of this kit can be measured by chemiluminescence instrument, microplate reader or liquid scintillation tester. The kit has the characteristics of rapid detection, high sensitivity, wide detection range and no interference of cell endogenous activity.Instruction：1.Cell lysis ( 1 ) Remove the medium and gently wash twice with PBS ( adherent cells can be operated directly, suspension cells need to be centrifuged to collect cells ). Add 1 × Lysis Buffer ( diluted component A with sterile water at 4 : 1 ) according to the following scheme, and then place the culture plate on a micro-oscillator at room temperature for 15 min to fully lyse the cells. Note : The pyrolysis products can be stored at room temperature for 6 h, and can be stored at − 70 °C for a long time ( the pyrolysis products cannot be repeatedly frozen and thawed ). ( 2 ) The pyrolysis products were centrifuged at 10000-15000 rpm for 3-5 min. After centrifugation, the supernatant was transferred into a new EP tube for subsequent detection. Note : Cells can be detected immediately after lysis, or frozen, and re-detected when needed. The frozen samples need to be thawed to room temperature for detection. 2. Preparation of working fluid ( 1 ) Restore all components to room temperature. ( 2 ) Dilute component C with component B to 0.2 mg / mL firefly luciferase working solution. Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the amount of a single experiment is small, it is recommended to be subpackaged into small specifications according to a single amount of use. ( 3 ) The E component was diluted into the renilla luciferase working solution with the D component, and the dilution method was 1 µL E component was added to the 49 µL D component. Note : Renilla luciferase working solution needs to be prepared now. 3.chemiluminescence value detection ( 1 ) According to the operation instructions of the instrument, the instrument with chemiluminescence detection function was opened, such as multifunctional microplate reader. The parameters were set, the determination time was 10 s, and the determination interval was 2 s. ( 2 ) each sample determination, take the sample 20-100 µL ( if the sample volume is enough, please add 100 µL ; if the sample amount is insufficient, the amount can be appropriately reduced, but the amount of detection holes needs to be consistent ). 1 × Lysis Buffer was blank control. ( 3 ) 100 µL firefly luciferase working solution was added to determine the RLU ( relative light unit ) value ( it is recommended that the microplate reader set up the Shaking mixing function ). Note : Since the luminescence is instantaneous, it is recommended to detect immediately after adding the firefly luciferase working solution. ( 4 ) 100 µL renilla luciferase working solution was added to determine the RLU ( relative light unit ) value ( Shaking mixing function is recommended for microplate reader ). ( 5 ) In the case of renilla luciferase as an internal reference, the RLU value measured by firefly luciferase was divided by the RLU value measured by renilla luciferase. According to the obtained ratio, the activation degree of the target reporter gene between different samples was compared. If firefly luciferase is used as an internal reference, similar calculations can also be performed.Component：Recommendation：It is recommended to use component B in advance to prepare 2 mg / mL storage solution, component B, component D and component C prepared as storage solution, and to carry out small batch packing according to the experimental requirements. All test working fluids are recommended to be used now to avoid repeated freezing and thawing.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. in order to obtain the best determination effect, when using a single tube chemiluminescence instrument for determination, the time from the mixing of sample and determination reagent to the pre determination should be controlled as much as possible; When using a multi-functional fluorescent microplate reader with chemiluminescence detection function, it is advisable to add all samples first, and then uniformly add firefly luciferase detection reagent. 3. the strongest wavelength of firefly luciferase catalyzed bioluminescence is 560 nm, and the strongest wavelength of Renilla luciferase catalyzed bioluminescence is 480 nm. 4. to prevent interference between holes, it is recommended to use white opaque orifice plate. 5. due to the influence of temperature on enzyme reaction, the sample and reagent should be measured after reaching room temperature. 6. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Study on gene expression regulation and promoter... 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 | Reverse transcriptases are enzymes encoded in retroviruses viral genome. The enzyme is responsible for transcription of the viral RNA to produce a dsDNA that can be inserted into the host genome.Reverse transcriptases are multifunctional enzymes. These enzymes exhibit an RNA and DNA directed Reverse transcriptases are enzymes encoded in retroviruses viral genome. The enzyme is responsible for transcription of the viral RNA to produce a dsDNA that can be inserted into the host genome.Reverse transcriptases are multifunctional enzymes. These enzymes exhibit an RNA and DNA directed polymerase activity. In addition reverse transcriptases catalyze the degradation of RNA in an RNA-DNA hybrid. The exonucleolytic activity proceeds in a 5' ---> 3' direction. The RNA or DNA directed activity requires a template (RNA or DNA) and a primer. The following is a schematic illustration of the reaction:Unit definition: One unit incorporates 1 nanomole of tritiated dTMP into acid insoluble productsusing poly(A)•oligo(dT) 12-18 as the template-primer in 20 minutes at 37° C.ApplicationsHIV reverse transcriptase is used for research on the AIDS primer. However it can be substituted for AMV reverse transcriptase, which is mainly used to transcribe mRNA into double stranded cDNA, that can be inserted into prokaryotic vectors. The enzyme can also be used with either single stranded DNA or RNA templates to make probes for use in hybridization experiments. It can be used for labeling the termini of DNA fragments with protruding 5' termini. The enzyme can also be used to sequence DNAs by the dideoxy chain termination method of Sanger when the Klenow fragment of E. coli DNA polymerase I, or the T7 DNA polymerase yield unsatisfactory results.Reagents0.05 M Tris, pH 8.3, containing 0.008 M MgCl21 mg/ml polyadenylic acid in water (poly A)DNA primer:Oligo d(T)12-181 µ mole dTTP/mL stock solution[methyl-3H]-Thymidine 5'-triphosphate (3H-dTTP)dTTP-3H-dTTP working mix: Add 1-2 µL 3H-dTTP per mL of 100 nmol/mL dTTP in order to obtain 1 to 1.5 x 105 cpm/mL1% bovine serum albumin10% perchloric acid1% perchloric acidBuffer substrate reaction mixture: Prepare fresh, immediately before use:For each 1mL of reaction mixture required mix:0.7 mL Tris/HCl, pH 8.3, 0.008M MgCl20.3 mL 1 mg/mL poly(A) RNA template0.005 mL 0.02 mg/mL oligo d(T)12-18 DNA primer0.02mL 1% BSAEnzymedilute as needed wtih 0.05M Tris/HCl, pH 8.3, 0.008M MgCl2 containing 0.1 mg/mL (1%) BSAProcedurePipette into each tube as follows:Buffer substrate mix：0.1 mLdTTP-3H3-dTTP：0.1 mLEnzyme：5-10 µLIncubate 20 minutes at 37° C. Stop reaction by adding 1 ml 10% cold perchloric acid. Filter through 0.2µ manifold filters used with Millipore vacuum manifold. Wash four times using 2mL 1% cold perchloric acid/wash. Transfer filter to scintillation vials. Add 2mL Cellosolve (or 2-methoxyethanol) to dissolve filter. Filters become opaque upon addition of Cellosolve. Make sure filters are dissolved before proceeding. Add 10mL scintillation cocktail and count.Calculation... Read More | 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 | Purity>97% by SDS-PAGE and HPLC analyses.FunctionPlays an important role in the organization of the cytoskeleton (By similarity). Binds to and sequesters actin monomers (G actin) and therefore inhibits actin polymerization. Seraspenide inhibits the entry of hematopoeitic pluripotent stem cells Purity>97% by SDS-PAGE and HPLC analyses.FunctionPlays an important role in the organization of the cytoskeleton (By similarity). Binds to and sequesters actin monomers (G actin) and therefore inhibits actin polymerization. Seraspenide inhibits the entry of hematopoeitic pluripotent stem cells into the S-phase... Read More |