| Description | Absorbance: A412 nm = 1.10 at 1/50 dilution in deionized waterGeneral DescriptionEr are rabbit red blood cells which have been washed free of rabbit plasmaproteins, but they are not coated with antibodies as is done with EA. These cells have traditionally been used as cells that spontaneously Absorbance: A412 nm = 1.10 at 1/50 dilution in deionized waterGeneral DescriptionEr are rabbit red blood cells which have been washed free of rabbit plasmaproteins, but they are not coated with antibodies as is done with EA. These cells have traditionally been used as cells that spontaneously activate the human alternative complement pathway in serum samples (Morgan, B.P. (2000; Dodds, A.W. and Sim, R.B. (1997)). Most human serum samples have small amounts of natural antibodies (usually IgG) to rabbit antigens and at high concentrations of serum these will agglutinate Er. This will activate the classical pathway if not blocked. Blocking the classical pathway is usually accomplished with 3 to 13 mM MgEGTA.Er are supplied at assay-ready concentrations in the traditional buffer used for APH50 assays (GVB°) which lacks metal ions. They can usually be used for 2 weeks after preparation. They are shipped cold, but are not harmed by extended periods at room temperature (note that they circulate 60+ days at 37℃in vivo). They should be washed once before each use (3-5 min at 500 to 1000 x g at 4℃) and resuspended in GVB° to reduce background and readjust their concentration. This procedure may also be used to concentrate the cells.Physical CharacteristicsEr are natural uncoated (non-opsonized) rabbit erythrocytes.AssaysAlthough the APH50 titer is widely used and serves as the primary test for alternative pathway complement activity, the APH50 assay procedure is not entirely standardized. In research labs there are as many procedures as there are labs, but all are basically similar and give useful results. The main difference between the classical pathway assays for CH50 determination and the alternative pathway assays for APH50 is the concentration of serum required. Alternative pathway activity becomes ineffective at dilutions of serum beyond 1/10 to 1/30. In contrast, CH50 titers are performed in the range 1/100 to 1/500 dilution. Dilution silences the alternative pathway in CH50 titers, but APH50 measurements would be overwhelmed by the classical pathway if this pathway were not blocked. Blocking of alternative pathway activity may be accomplished by using serum depleted of factor B or D or most conveniently by adding 3 to 13 mM MgEGTA (final concentration) to the assays which completely inhibits the classical pathway by chelating calcium ions and dissociating C1. Briefly, a complement-containing serum sample is diluted such that the final dilutions in the assay are in the range from 1/2 to 1/20. Controls include two tubes with no complement for the 0% lysis background and two tubes for 100% lysis. Assay tubes should be set up on ice. The assays are performed in 200 µL containing 4 to 60 µL of test serum, 10 µL 0.1 M MgEGTA, GVB°, and 50 µL Er. The assays are mixed and placed in a 37℃ water bath with remixing at approximately 10 min intervals. After 30 min, 2 mL of cold GVBE is added, mixed and the unlysed cells are spun down (500-1000 x g for 3 min). The absorbance of the supernatant should be determined at 412 nm in a 1 cm cuvette and the percentage of specific lysis calculated after subtracting the background and dividing by the 100% lysis control. The data is plotted as percent lysis on the y axis and µL serum on the x axis. The APH50 value of the complement sample is calculated from the amount of serum needed to cause lysis of 50% of the cells. For example, if it takes 12 µL NHS to lyse 50% of the cells then this serum would contain 83 AHP50 units/mL (1000 µL /12 µL = 83 units/mL). It should be noted that somewhat different APH50 values can be obtained on what should be identical samples. This is the result of the vast number of variables involved in APH50 determinations including the fact that each batch of rabbit erythrocytes is slightly different.ApplicationsEr cells are primarily used to determine the activity of the alternative pathway of complement (APH50 titer) (Morgan, B.P. (2000; Dodds, A.W. and Sim, R.B. (1997)). Natural antibodies present in human blood to animal antigens may cause agglutination of the cells. This antibody may also cause lysis if the classical pathway is not blocked.Rabbit erythrocytes are attacked and lysed by the alternative pathways of most mammalian species. It has not been clearly demonstrated why rabbit complement does not lyse rabbit Er when most other animal species do lyse these cells, although they do carry rabbit DAF which works on rabbit, but not human complement enzymes.RegulationRabbit erythrocytes (Er) are used for human complement assays partly for convenience, but also because they lack membrane-bound regulators of human complement. No significant level of functional DAF, CD59 or CR1 exists on Er for human complement. Thus, Er are useful for their lack of membrane regulatory activities... Read More | Purity：>95%(SDS-PAGE) Function：Cooperates with MD-2 and TLR4 to mediate the innate immune response to bacterial lipopolysaccharide (LPS). Acts via MyD88, TIRAP and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. Up-regulates cell surface Purity：>95%(SDS-PAGE) Function：Cooperates with MD-2 and TLR4 to mediate the innate immune response to bacterial lipopolysaccharide (LPS). Acts via MyD88, TIRAP and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. Up-regulates cell surface molecules, including adhesion molecules.Background：CD14 is a 55 kDa cell surface glycoprotein that is preferentially expressed on monocytes/macrophages. The human CD14 cDNA encodes a 375 amino acid (aa) residue precursor protein with a 19 aa signal peptide and a C-terminal hydrophobic region characteristic for glycosylphosphatidyinositol (GPI)-anchored proteins. Human CD14 has four potential N-linked glycosylation sites and also bears O-linked carbohydrates. The amino acid sequence of human CD14 is approximately 65% identical with the mouse, rat, rabbit, and bovine proteins. CD14 is a pattern recognition receptor that binds lipopolysaccharides (LPS) and a variety of ligands derived from different microbial sources. The binding of CD14 with LPS is catalyzed by LPS-binding protein (LBP). The toll-like-receptors have also been implicated in the transduction of CD14-LPS signals. Similar to other GPI-anchored proteins, soluble CD14 can be released from the cell surface by phosphatidyinositol-specific phospholipase C. Soluble CD14 has been detected in serum and body fluids. High concentrations of soluble CD14 have been shown to inhibit LPS-mediated responses. However, soluble CD14 can also potentiate LPS response in cells that do not express cell surface CD14... Read More | Purity>97% SDS-PAGE and HPLC analyses. FunctionLA-PF4 stimulates DNA synthesis, mitosis, glycolysis, intracellular cAMP accumulation, prostaglandin E2 secretion, and synthesis of hyaluronic acid and sulfated glycosaminoglycan. It also stimulates the formation and secretion of plasminogen Purity>97% SDS-PAGE and HPLC analyses. FunctionLA-PF4 stimulates DNA synthesis, mitosis, glycolysis, intracellular cAMP accumulation, prostaglandin E2 secretion, and synthesis of hyaluronic acid and sulfated glycosaminoglycan. It also stimulates the formation and secretion of plasminogen activator by human synovial cells. NAP-2 is a ligand for CXCR1 and CXCR2, and NAP-2, NAP-2(73), NAP-2(74), NAP-2(1-66), and most potent NAP-2(1-63) are chemoattractants and activators for neutrophils. TC-1 and TC-2 are antibacterial proteins, in vitro released from activated platelet alpha-granules. CTAP-III(1-81) is more potent than CTAP-III desensitize chemokine-induced neutrophil activation.Post-translationalProteolytic removal of residues 1-9 produces the active peptide connective tissue-activating peptide III (CTAP-III) (low-affinity platelet factor IV (LA-PF4)). Proteolytic removal of residues 1-13 produces the active peptide beta-thromboglobulin, which is released from platelets along with platelet factor 4 and platelet-derived growth factor. NAP-2(1-66) is produced by proteolytical processing, probably after secretion by leukocytes other than neutrophils. NAP-2(73) and NAP-2(74) seem not be produced by proteolytical processing of secreted precursors but are released in an active form from platelets... Read More | Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description:Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), which is a member of orthopoxvirus genus. A35R gene is highly conserved among poxviruses and encodes a previously uncharacterized hydrophobic acidicPurity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description:Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), which is a member of orthopoxvirus genus. A35R gene is highly conserved among poxviruses and encodes a previously uncharacterized hydrophobic acidic protein. The A35R has little homology to any protein outside of poxviruses, suggesting a novel virulence Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), which is a member of orthopoxvirus genus. A35R gene is highly conserved among poxviruses and encodes a previously uncharacterized hydrophobic acidic protein. The A35R has little homology to any protein outside of poxviruses, suggesting a novel virulence mechanism.A35R could block some stage of antigen processing or presentation in infected cells or interfere with regulation of apoptosis. In addition, the A35R function may be required for growth in certain cell types, e.g., macrophage, in vivo. It localizes to factories where viral DNA is located and it was shown to be a constitutive transcriptional activator in a large-scale yeast two-hybrid study... Read More | BackgroundStreptavidin is a tetrameric bacterial protein isolated from Streptomyces avidinii providing 4 high-affinity biotin binding sites. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin. With a dissociation constant on the order of ≈10⁻¹⁴ mol/L,BackgroundStreptavidin is a tetrameric bacterial protein isolated from Streptomyces avidinii providing 4 high-affinity biotin binding sites. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin. With a dissociation constant on the order of ≈10⁻¹⁴ mol/L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. Unlike egg-white avidin, which has a net positive charge at neutral pH and contains about 7% carbohydrate, streptavidin has almost no net charge at neutral pH, does not contain carbohydrate, and exhibits lower non-specific background. Streptavidin conjugates are widely used together with a conjugate of biotin for specific detection of a variety of proteins, protein motifs, nucleic acids and other molecules. This FITC-streptavidin conjugate was prepared by highly purified Streptavidin and free FITC was removed. Streptavidin (FITC) is a useful second-step reagent for the indirect immunofluorescent staining of cells in combination with biotinylated primary antibodies for flow cytometric analysis. Excitation at 488nm light leads to a fluorescence emission maximum of 520 nm.Recommended Usage：Every lot of Streptavidin-FITC is tested by flow cytometry using biotinylated primary antibodies. From this testing it is recommended that between 0.02 and 0.25 µg of streptavidin be used per 106 cells in a 100 µl staining volume... Read More |