| Description | Purity >90% by SDS-PAGEExtinction CoeffA280 nm = 1.04 at 1.0 mg/mLPrecautionsUse normal precautions for handling human blood products.General DescriptionNative human C7 is a naturally glycosylated protein composed of a single polypeptide chain of 92,400 Da. C7 is essential for formation of the Purity >90% by SDS-PAGEExtinction CoeffA280 nm = 1.04 at 1.0 mg/mLPrecautionsUse normal precautions for handling human blood products.General DescriptionNative human C7 is a naturally glycosylated protein composed of a single polypeptide chain of 92,400 Da. C7 is essential for formation of the membrane attack complex (MAC) and is activated non-proteolytically by binding to recently-formed C5b,C6 complexes at the cell membrane. Each pathway of complement activation generates proteolytic enzyme complexes (C3/C5 convertases) which are bound to the target surface (Ross, G.D. (1986); Law, S.K.A. and Reid, K.B.M. (1995)). 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 surroundingfluid 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. This complex binds C9 and each bound C9 can bind another C9 initiating formation of a ring structure containing up to 18 C9 molecules (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-8 complex. 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 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 approximately 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 C7 is 92,400 Da and it is composed of a single polypeptide chain. The pI of C7 is heterogeneous from 6.0 to 6.5.FunctionSee General Description above.AssaysThe simplest assay for C7 is to use C7-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 C7. Each unique application might require appropriate conditions to be determined. However, a typical assay would involve mixing on wet ice 25 µL C7-Dpl, C7-containing sample diluted with GVB⁺⁺ to contain from 0.5 to 5 ng C7, 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 C7 or normal human serum (NHS) may be used as a source of C7. The reaction mixture is incubated for 30 min at 37℃ and 1 mL of cold GVBE added, mixed and centrifuged to spin out unlysed cells. The released hemoglobin in the supernatant is then analyzed at 415 nm and compared to blanks without C5 (background lysis control) and cells incubated with 275 µL water in place of GVB++ and 25 µL C7-Dpl (100% lysis control).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 above.In vivoThe normal serum concentration of C7 is 56 µg/mL (normal range 49 to 70µg/mL). The primary site of synthesis is the liver. C7 is a weak acute phase protein and its synthesis is stimulated by the cytokines 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 C7 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 that binds to C5b-9 complexes that fail to insert in the target cell membrane. This reduces damage to nearby host cells. 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 do 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 inactivate C3/C5 convertases and the ability to inactivate C5b-9. This results in complement-mediated damage to and eventual lysis of long-lived blood cells such as erythrocytes and platelets.Genetics Human chromosome location 5q 12-14. Accession number HSC6A. Mouse chromosome 15. Human genomic structure: the gene spans 80 kb with 18 exons.DeficienciesHuman deficiencies have been found and 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 by certified 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 | Inquire | Inquire | Inquire | 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 |