| Description | LL-37, human TFA is a 37-residue, amphipathic, cathelicidin-derived antimicrobial peptide, which exhibits a broad spectrum of antimicrobial activity. LL-37, human TFA could help protect the cornea from infection and modulates wound healing | Inquire | Purity>90% by SDS-PAGEExtinction Coeff.A280 nm = 0.988 at 1.0 mg/mLPrecautionsUse normal precautions for handling human blood productsGeneral DescriptionNative human C9 is a naturally glycosylated (7.8%) protein composed of a singlepolypeptide chain. The molecular weight is 71,000 Da. C9 binds toPurity>90% by SDS-PAGEExtinction Coeff.A280 nm = 0.988 at 1.0 mg/mLPrecautionsUse normal precautions for handling human blood productsGeneral DescriptionNative human C9 is a naturally glycosylated (7.8%) protein composed of a singlepolypeptide chain. The molecular weight is 71,000 Da. C9 binds to the C5b-8 complex and forms the mature membrane attack complex (MAC) on cell membranes. Each pathway of complement activation generates proteolytic enzyme complexes (C3/C5 convertases) which are bound to the target surface (Ross, G.D. (1986)). These enzymes cleave a peptide bond in the larger alpha chain of C5 releasing the anaphylatoxin C5a and activating C5b. This is the only proteolytic step in the assembly of the C5b-9 complex. C5b is unstable, but it remains bound to the activating complex for a brief time (~2 min) during which it either binds a single C6 from the surrounding fluid or decays and is no longer capable of forming MAC. The C5b,6 complex may also remain bound to the C3/C5 convertase where the binding of a single C7 exposes a membrane-binding region and C5b,6,7 can partially insert into the bilipid layer of the target cell. Up to this point the complex may diffuse away from the target cell and enter the membrane of a nearby cell. This is called bystander lysis or “reactive lysis” and can be a significant source of pathology. Each C5b-7 complex can bind one C8 protein molecule which results in the complex inserting more firmly into the membrane. The C5b-8 complex is capable of causing lysis without C9 although this is slow and requires many more complexes per cell than with C9. This property complicates C9 titrations since the precursor (C5b-8) can also cause lysis. The primary role of C8 is to catalyze the binding of C9 and each bound C9 can bind another C9 initiating formation of a ring structure containing up to 18 molecules of C9 (Podack, E.R. (1984)). C5b-9 complexes with one or more C9 are referred to as the Membrane Attack Complex (MAC) of complement. Not all C5b-8 complexes have complete rings of C9 with the average being only three C9 per C5b-8complex. Nevertheless, these structures are capable of causing lysis if enough are formed in a given cell. Completed protein rings of C9 form the pores seen on electron micrographs and they result in leakage of metabolites and small proteins out of the cell as well as movement of water into the cell. If sufficient numbers are inserted into a cell membrane then water flowing into the cell, due to osmotic pressure, will rupture the cell membrane allowing the entire contents of the target cell (or a bystander cell) to be released. Either process may result in cell death. Originally it was thought that this required only one C5b-9 complex per cell (referred to as the “one hit theory” of lysis (Rommel F.A. and Mayer, M.M. (1973)), but this is probably not correct. For example, an erythrocyte without CD59 requires ~850 C5b-9 complexes, as measured by the number of C7 molecules, for lysis to occur (Bauer, J. et al. (1979)). Host cells protected from MAC by CD59 require sufficient numbers of C5b-9 to tie up all the CD59 and then ~850 C5b-9 in addition. Lysis of nucleated cells requires many more C5b-9 complexes due to their size and due to the presence of multiple defense mechanisms in such cells.Physical Characteristics & StructureThe molecular weight of C9 is 71,000 Da and it is a single polypeptide chain. The protein contains 7.8% carbohydrate attached at two N-linked glycosylation sites. The pI of C9 is 4.7. C9 may polymerize spontaneously forming MAC rings without C5b-8. The rings formed from pure C9 as well as the completed rings formed by C5b-9 with 12 to 18 C9 molecules have the unusual property of being stable in boiling SDS even though they are non-covalently bound. Function See General Description above. Assays Assays for C9 function are complicated by the fact that if excess C5-C8 is used cells (EA or Er) will be lysed by the C5b-8 complex. Thus it is critical to use limited C8 in these assays to keep the background lysis to a minimum. The simplest assay for C9 is to use C9-depleted human serum and measure the lysis of EA (classical pathway) or Er (alternative pathway) as a function of the concentration of added test sample or standard purified C9. Each unique application might require appropriate conditions to be determined. However, a typical assay would involve mixing on wet ice ~5 µL C9-Dpl, C9-containing sample diluted with GVB⁺⁺ to contain from 1 to 10 ng C9, and sufficient GVB⁺⁺ to bring the volume to 300 µL. EA (3 X 10⁷ cells in 200 µL) diluted in GVB⁺⁺ should be added last. Purified C9 or normal human serum (NHS) may be used as a source of C9. The reaction mixture is incubated for 30 min at 37℃ and 1 mL of cold GVBE added, mixed and centrifuged to spin down unlysed cells. The released hemoglobin in the supernatant is then analyzed at 415 nm and compared to blanks without C9 (background lysis control) and cells incubated with 275 µL water instead of GVB⁺⁺ and 25 µL C9-Dpl (100% lysis control). Note as mentioned above, at inputs of serum higher than ~5 µL of C9-Dpl, EA and other target cells may also be lysed in the absence of C9 depending on the cells’ susceptibility to C5b-9.Many other assays have been described using EA preloaded with C1 (EAC1 cells) or preloaded with the classical pathway C5 convertase (EAC1423 cells), however, all these assays require the use of multiple purified complement components or more difficult-to-prepare reagents (Dodds, A.W. and Sim, R.B. (1997; Morgan, B.P. (2000);Tack, B.F., et al. (1981)).ApplicationsSee General Description aboveIn vivoThe normal serum concentration of C9 is 60 µg/mL (normal range 47 to 70µg/mL). The primary site of synthesis is the liver. C9 is also produced by monocytes, macrophages, fibroblasts and glial cells. C9 is an acute phase protein and its synthesis is stimulated by cytokines (such as IFNγ) that stimulate increased biosynthesis of many other complement proteins.RegulationMany proteins and other components of plasma have an inhibitory effect on the lytic activity of C5b-9 complexes but there are no specific C9 inactivators. Most of the C5b-9 inhibitors interact with the complex after the C5b-7 stage. If any of the C5bcontaining complexes fail to insert into a membrane they may self-aggregate or bind to regulatory proteins the most prevalent of which is S Protein. S Protein (also called vitronectin) is an 80,000 Da plasma protein found bound to most soluble C5b-9 complexes. Many other serum components inhibit or partially inhibit lysis by C5b-9 and these include SP40,40 (also known as clusterin and apolipoprotein J) and many plasma lipoprotein complexes (LDL, HDL, etc.).Host cells protect themselves from C5b-9 by a variety of mechanisms. Membrane proteins DAF, MCP, and CR1 inhibit formation of C3/C5 convertases preventing MAC formation. CD59, also called “homologous restriction factor” and “protectin”, is a 18,000 to 20,000 Da ubiquitous component of cell membranes that is very effective at binding to and inhibiting the lytic potential of C5b-8 and C5b-9 complexes. The speciesspecificity of CD59 is not absolute and many mammalian CD59 proteins inhibit or partially inhibit MAC from other species. The specificity that is observed appears to be due to incompatibilities between C8 of one animal and the CD59 of another. Like DAF, CD59 contains a GPI anchor (a post-translationally added lipid tail that inserts into the bilipid layer of the cell). The disease PNH is caused by the loss of enzymes that attach the GPI tail, thus depriving cells of the ability to express DAF and inactivate C3/C5 convertases and the ability express CD59 to inactivate C5b-9. This results in the spontaneous lysis by complement of the most susceptible cells such as erythrocytes and platelets.GeneticsHuman chromosome location 5p 13. Accession number HSC6A. Mouse chromosome 15. Human genomic structure: the gene spans 100 kb with 11 exons.DeficienciesHuman C9 deficiencies are quite common. A well documented study found that 1:1000 people in the Janaese population were C9 deficient although other ethnic groups have lower incidents of C9 deficiency (Horiuchi, T. et al. (1998)). Deficiencies exhibit autosomal recessive transmission. Patients generally exhibit abnormally high susceptibility to recurrent meningococcal meningitis and systemic neisserial infections. Partial deficiencies do not seem to show adverse clinical effects.DiseasesSee Deficiencies above.Precautions/Toxicity/HazardsThis protein is purified from human plasma, therefore precautions appropriate for handling any blood-derived product must be used even though the source was shown bycertified tests to be negative for HBsAg, HTLV-I/II, STS, and for antibodies to HCV, HIV-1 and HIV-II.Hazard Code: B WGK Germany 3MSDS available upon request... Read More | Product contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a preProduct contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, including Fast Taq DNA Polymerase, PCR Buffer, dNTPs, Mg2+ and so on, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequence and cDNA target sequence after RNA reverse transcription. The Fast Taq DNA Polymerase contained in this product can effectively reduce the non-specific amplification generated by the non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme only needs to be incubated at 95 ℃ for 30 s. The whole PCR reaction process can save about 40 minutes compared with the ordinary reaction, which greatly shortens the reaction time of PCR. The combination of unique PCR buffer system and fast hot start enzyme effectively inhibits the generation of non-specific products and significantly improves the PCR amplification efficiency with stronger fluorescence signal, higher sensitivity and wider linear range. The product has a wide range of applications and can be used for both normal and rapid quantitative PCR programs.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration (F665766):Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments that require Low ROX calibration (F665768):ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments that require High ROX calibration (F665774):ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.2. Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.UsageThe following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction systemreagents50µl reaction systemfinal concentration2×Fast Probe Mixture25 µl1×Forward Primer, 10µM1µl0.2µM¹⁾Reverse Primer, 10µM1µl0.2µM¹⁾Probe, 10 µM1µl0.2µM²⁾Template DNA2µl³⁾ 50x Low ROX or High ROX(optional)⁴⁾1µl1×ddH₂Oup to 50µlNote: 1) Usually the primer concentration of 0.2µM can get better results, and 0.1-1.0µM can be used as a reference for setting the range. 2) The final 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 instruction manual of the instrument or the specific requirements of the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.2. PCR reaction program:A two-step PCR reaction program is recommended, and this program is set up using the ABI 7500 Fluorescent Quantitative PCR Instrument as a reference.Note: 1) The enzyme used in this product must be pre-denatured at 95°C for 30s to achieve enzyme activation. Under this condition, most of the templates can be well unchained. For templates with high GC content and complex secondary structure, the pre-denaturation time can be extended to 1-4 minutes to allow the starting template to fully unchain.(2) It is recommended to use two-step PCR reaction program, if you do not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out three-step PCR amplification, and the annealing temperature, please use the range of 56 ℃ - 64 ℃ as a setting reference... Read More | Biochemical Test:SDS-PAGE (purity > 80%); Western blot with patient sample.Calculated Isoelectric Point:pH 6.64 |