| Description | ALMS1 Human Pre-designed siRNA Set A contains three designed siRNAs for ALMS1 gene (Human), as well as a negative control, a positive control, and a FAM-labeled negative control. Components ALMS1 siRNA-1: 5 nmol (HPLC) ALMS1 siRNA-2: 5 nmol (HPLC) ALMS1 siRNA-3: 5 nmol (HPLC) siRNA Negative Control:ALMS1 Human Pre-designed siRNA Set A contains three designed siRNAs for ALMS1 gene (Human), as well as a negative control, a positive control, and a FAM-labeled negative control. Components ALMS1 siRNA-1: 5 nmol (HPLC) ALMS1 siRNA-2: 5 nmol (HPLC) ALMS1 siRNA-3: 5 nmol (HPLC) siRNA Negative Control: 5 nmol (HPLC) FAM-labeled siRNA Negative Control: 5 nmol (HPLC) GAPDH siRNA Positive Control:5 nmol (HPLC)... Read More | Biochemical Test:SDS-PAGE (purity > 80%); Western blot with patient sample.Calculated Isoelectric Point:pH 6.47 | Protein Purity>97% by SDS-PAGEExtinction Coeff.A280 nm = 0.41 at 1.0 mg/mlMolecular Weight10,400 Da (single chain)General DescriptionNatural human C5a is prepared from human C5 protein by cleavage of the peptide bond between C5a and C5b by the human C5 convertase. C5a is a naturally glycosylated Protein Purity>97% by SDS-PAGEExtinction Coeff.A280 nm = 0.41 at 1.0 mg/mlMolecular Weight10,400 Da (single chain)General DescriptionNatural human C5a is prepared from human C5 protein by cleavage of the peptide bond between C5a and C5b by the human C5 convertase. C5a is a naturally glycosylated polypeptide containing 74 amino acids with a molecular weight of approx. 10,400 daltons. Itcontains 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). Its biological properties include being strongly chemotactic for neutrophils (PMN), causing smooth muscle contraction, increasing vascular permeability, causing histamine and TNFalpha release, and causing lysosomal degranulation of immune cells. C5a acts 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 non-immunolgical functions in organ development, CNS development, neurodegeneration, tissue regeneration and hematopoiesis (Monk, P.N. et al. (2007)).Native versus Recombinant C5aNumerous recombinant forms of C5a are sold by many companies. In side-by-side biological testing, we have found that our native C5a is 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 & StructureMolecular weight: 10,400 (+ 1,000 due to variable glycosylation)Deglycosylated MW: 8,271 (observed). Calculated monoisotopic mass 8268;Calculated average mass 8273.Isoelectric point: pI = 8.9Carbohydrate content: ~25% carbohydrate (heterogeneous) Amino acid sequence: TLQKKIEEIA AKYKHSVVKK CCYDGACVNNDETCEQRAAR ISLGPRCIKA FTECCVVASQ LRANISHKDM QLGRCAS Number: 80295-54-1MDL Number: MFCD00130842NMRderived structure: FEBS Lett. 238:289-294, 1988; Biochemistry 28:172-185,1989; Biochemistry 29:2895-2905, 1990; Proteins 28:261-267, 1997.FunctionC5a released from C5 by C5 convertases initiates a multitude of inflammatory reactions. C5a causes neutrophils to become adherent to endothelium and to migrate to the site of complement activation by chemotaxis where it stimulates release of PMN granule contents and reactive oxygen species. The biological properties of C5a include being strongly chemotactic for neutrophils (PMN), causing smooth muscle contraction, increasingvascular permeability, causing histamine release, and initiating lysosomal degranulation of a variety of immune cells. C5a acts through the C5a Receptor (C5aR, a G-protein coupled receptor) on PMN, monocytes, alveolar macrophages, dendritic cells, mast cells, glial cells and smooth muscle cells. Rapid release of C5a and other anaphylatoxins can cause systemic effects as well as local changes. Anaphylatic shock is a generalized circulatory collapse similar to that caused by an allergic reaction and is caused by C3a and C5a which are generally released together. AssaysThe multitude of biological functions of C5a has resulted in the use of many different assay systems (Dodds, A.W. and Sim, R.B. (1997)). 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. In addition, assays have been described that measure ATP release from guinea pig platelets, serotonin relaease from guinea pig platelets, N-acetyl-beta-D-glucosamidase release from differentiated U937 cells and calcium release from differentiated U937 cells. These assays have been described in detail (Dodds, A.W. and Sim, R.B. (1997)). Functional responses have been detected in the sub-picomolar concentration range for purified human C5a (Gerard, C. et al. (1981); Hugli, T.E. et al. (1981)).ELISA kits for the assay of C5a levels (or more correctly C5a desArg levels) in blood and other fluids are sold by many companies. A radioimmunoassay for C5a/C5a desArg is also available. 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 has been reported to be approximately 4 nMalthough it is difficult to draw or store 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.RegulationC5a levels are regulated by three processes: formation, inactivation 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 is “inactivated” by removal of its Cterminal arginine amino acid. The product C5a desArg (or C5a without the C-terminal arginine) is produced by the action of the plasma enzyme carboxypeptidase N (Mueller-Ortiz S.L. et al. (2009)). 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 results in its rapid removal from circulation.DeficienciesA deficiency of C5 or a deficiency of the enzymes that cleave C5 to generate C5a result in the absence of C5a. 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. Humans lacking C5 are susceptible to repeated infections from a wide variety of organisms, primarily gramnegative 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 the absence of C5a is unclear, but information on this is being 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 3... Read More | ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology experiments, such as PCR, enzyme digestion, hybridization, microarray analysis, and other molecular biology experiments.The product was quantified using NanoDrop One at a concentration of 200 ng/µL.Preparation and precautions before useLong-term storage at -20˚C is recommended. Before use, the bottle should be removed from the refrigerator and equilibrated to room temperature and centrifuged before opening the cap for use. Samples should be restored to the sealed state as soon as possible after opening.How to use (take qPCR experiment as an example)1. Amplification template preparationThe samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0,1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.05-10 ng/µL. The samples were placed on ice at 4°C and set aside.2. Standard dilution: according to the following table, firstly dilute Human DNA Standard 1 (100ng/uL) with TE to make 5 different concentrations of standards according to the table below. 10ng/µL of DNA Standard 1 (Std. 1) can be stored stably at -20℃ for 1 month; Std2-5 can only be used on the same day, and should be placed at 4℃ or on ice when not in use for the time being after preparation. When not used temporarily after preparation, it should be stored at 4℃ or on ice.styleCorresponding concentration (ng/µL)Minimum dilution volume (in µL)Std.11010 [100 ng/µL DNA Standard 1] + 90 TEStd.22.520 [Std. 1] +60 TEStd.30.62520 [Std. 2] +60 TEStd.40.1562520 [Std. 3] +60 TEStd.50.039062520 [Std. 4] +60 TE3. qPCR reaction system preparationThe cryopreserved reagents to be used were completely thawed and mixed by inversion several times before preparation, and then briefly centrifuged and prepared for use. 20 µL of the base reaction system was as follows.The base reaction system for 20 µL was as follows:reagents20µL reaction system2×qPCRMix10µLPrimerMixXµLProbeMixXµLTemplate4µLddH2OMake up to 20 µLNote: High Rox model: add 1 µL of 50×High Rox per 50 µL of reaction system; Low Rox model: add 1 µL of 50×High Rox per 500 µL of reaction system.Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.The concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the manual of the instrument or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration during actual use.Prepare a sufficient amount of reaction system mixture as required. After the reaction system has been prepared and mixed thoroughly, add 16 µL per well to the reaction wells. Then add the prepared standard and diluted sample into the corresponding reaction wells, the volume of addition is 4µL/well. TE was added to the blank control tube, and the same amount of TE was added at 4 µL/well.It is recommended to use 20 µL for the reaction, if you need to perform a smaller system reaction, reduce the system components in equal proportion.4. qPCR reaction programThe following is an example of our GoldStar Probe Mixture reaction conditions, which should be improved and optimized according to the PCR product template, primer structure and target fragment size.movetemptimingcirculatepremutability95°C10min1denaturation95°C10sec55Annealing/Extension60°C30sec5Data analysis1. Standard curve productionThe standard curve was plotted with reference to the Excel sheet for data processing. The correlation coefficient R2 of the standard curve should not be lower than 0.98, and the slope should be between -3.1 and -3.6 when the Ct value is the vertical coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment... Read More | Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.This protein is one of the nuclear-coded polypeptide chains of cytochrome c oxidase, the terminal oxidase in mitochondrial electron transport |