| Description | Protein Purity>90% by SDS PAGEExtinction CoeffA280 nm = 1.682 at 1.0 mg/mlMolecular Weight155,000 Da (single chain)General DescriptionRat (Rattus Norvegicus) Complement factor H (fH) is purified from normal ratserum. Factor H is an essential regulatory component of the alternative pathway of Protein Purity>90% by SDS PAGEExtinction CoeffA280 nm = 1.682 at 1.0 mg/mlMolecular Weight155,000 Da (single chain)General DescriptionRat (Rattus Norvegicus) Complement factor H (fH) is purified from normal ratserum. Factor H is an essential regulatory component of the alternative pathway of complement. It is critical for prevention of complement activation on host cells and tissues, especially the kidney. It has two functional activities: 1) it controls the formation and decay of thealternative pathway C3/C5 convertase (decay accelerating activity) and 2) it acts as a cofactor for factor I which proteolytically inactivates C3b when C3b is bound to factor H (cofactor activity). A C3b-binding protein, similar to factor H isolated from rat plasma, has been reported to be produced by rat platelets and functions as an immune adherence receptor for clearance of immune complexes in rodents (Alexander J.J. et al. (2001)).Factor H is a 155,000 Da protein composed of 20 homologous domains arranged like beads on a semi-flexible string. The N-terminal 5 domains bind to C3b and inhibit binding of factor B thus reducing the formation of C3/C5 convertase. Factor H also binds to preformed C3/C5 convertases (C3b,Bb and C3b,Bb,C3b) and causes rapid release of the catalytic subunit Bb (decay acceleration). These activities are essential for controlling the spontaneous activation of the alternative pathway amplification process in plasma. In addition, factor H controls the formation and decay of these enzymes when C3b is attached to the surface of particles. It is most effective on host cells and less effective on foreign particles for reasons described below. The alternative pathway of complement is constantly activating by “tickover” producing fluid phase C3b-like C3(H2O) and C3b. Factor H can bind to these proteins and act as a cofactor so that factor I (a serine protease that circulates in active form) can cleave their alpha chains producing inactive proteins (iC3b or iC3(H2O)). If C3b is not inactivated in this way it continues to form C3 convertases and consumes factor B and C3. If C3b is attached to surfaces it is converted to iC3b by factors H and I in a similar manner. Factor H is more effective when C3b resides on a host cell due to the presence of host markers recognized by factor H. Complement-mediated damage to the host is minimized due to host specific recognition by factor H.Factor H appears to regulate discrimination between potential pathogens and host cells and tissues by recognizing host markers. C3b attached to a surface can initiate the amplification cascade of the alternative pathway. Factor H prevents this on host cells and allows it to occur on surfaces that do not bear host-like markers. These host-specific structures are thought to be polyanionic clusters such as sialic acids and sulfated glycosoaminoglycans. Recognition of host markers occurs through multiple polyanion binding sites located in domains 6-20 of factor H. One site is located in domain 7 and a mutation in this domain (Y402H) is strongly associated with complement activation and tissue destruction in age-related macular degeneration (Zipfel, P.F. et al. (2006)). A tentative site is located in the domain 12-14 region and a very important site is located at the C-terminal in domains 19-20. This C-terminal site appears to be the main site that aids binding to host surfaces. Mutations affecting or located in these domains lead to activation of the alternative pathway of complement in inherited hemolytic uremic syndrome (Zipfel, P.F. et al. (2006)). This site appears to be the site involved in polyanion-dependent dimer and tetramer formation of factor H (Pangburn, M.K. et al. (2009)).Physical Characteristics & StructureThe molecular weight of rat factor H has been reported to be about 150,000 to 155,000 daltons (Daha MR et al (1982); Demberg T et al., (2002); Alexander JJ et al., 2001)). Rat factor H is 9.5% glycosylated (Demberg T et al., (2002). Analysis of purified rat Factor H by SDS/polyacrylamide gel electrophoresis (Invitrogen) under non-reduced and reduced conditions shows a single band that migrates slightly ahead of human factor H (155,000 daltons). The extinction coefficient of rat Factor H (E1%/280nm = 16.82) is calculated based on its amino acid sequence using ProtParam and assumes all pairs of Cys residues form cystines (i.e. a pair of cysteine molecules are joined by a disulfide bond). The calculated pI based on its amino acid sequence is 6.29. The normal plasma concentration of Factor H rat serum has been reported to be 238 + 21ug/ml by Demberg T et al., (2002) while Daha MR et al (1982) have reported 244 + 21 ug/ml.FunctionSee General Description above.AssaysFunctional assays of factor H measure either its decay accelerating activity or its factor I cofactor activity (Morgan, B.P. (2000)). A continuously monitored fluorescent assay has been reported (Pangburn, M.K. et al. (1983)) which takes advantage of the approximately 8-fold drop in fluorescence of ANS (8-anilino-1-naphthalenesulfonic acid) in the presence of C3b when that C3b is converted to iC3b. Other functional assays of Factor H are described under the Assay section for human factor H. The cofactor activity of purified rat factor H was determined using the convenient cofactor assay that measures the cleavage of purified C3b by SDS gels. Four micrograms (4ug) of rat C3b was incubating with various amounts of rat factor H ranging from 0.1 to 1µg in the presence of 1µg human factor I in a total volume of 12 µL. The assays were set up on wet ice, then incubated for 15 min at 37oC at which time SDS sample buffer containing reducing agent were added to the tubes and the samples heated for 5 min. Analysis of SDS gels revealed > 90% cleavage of the alpha chain of rat C3b in the presence of > 0.02 ug rat factor H and 1 ug human factor I. FunctionThe biological functions of factor H are described above in the General Description section.GeneticsRat factor H chromosome location 13. The NCBI Gene ID number for rat factor H: 155012 and UniProt accession number is Q91YB6.Precautions/Toxicity/HazardsThis protein is purified from animal plasma/serum and therefore precautions appropriate for handling any animal blood-derived product must be used.Hazard Code: B WGK Germany 3MSDS available upon request.ReferencesMorgan, B.P. ed. (2000) Complement Methods and Protocols. (ISBN 0-89603-654-5) Humana Press, Inc., Totowa, New Jersey.Pangburn, M.K. and Müller-Eberhard, H.J. (1983) Kinetic and thermodynamic analysis of the control of C3b by the complement regulatory proteins factors H and I.Biochemistry 22:178-185.Pangburn, M.K., Rawal, N., Cortes, C., Alam, M.N., Ferreira, V.P. and Atkinson, M.A. (2009) Polyanion-induced self-association of complement factor H. J. Immunol. 182:10611068.Zipfel, P.F., Heinen, S., Jozsi, M. and Skerka, C. (2006) Complement and diseases: defective alternative pathway control results in kidney and eye diseases. Mol. Immunol. 43:97-106.Demberg, T., Pollok-Kopp B, Gerke D, Gotze O. and Schlaf G. (2002) Rat complement factor H: molecular cloning, sequencing and quantification with a newly established ELISA. Scand. J. Immunol. 56:149-160.Daha MR and van Es LA. (1982) Isolation, characterization and mechanism of action of rat β1H. J. Immunol. 128: 1839-1843.Alexander JJ, Hack BA, Cunningham PN and Quigg RJ. (2001) A Protein with characteristics of factor H is present on rodent platelets and functions as the immune adherence receptor. J. Biol. Chem. 276: 32129–32135... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Unit Definition One unit will cause a change in A600 of 0.330 per minute at pH 5.7 at 37°C in a 2.0 ml reaction mixture (45 minute assay) | This reagent kit is designed based on the principle that biotin and Streptavidin have a strong affinity. After the primary antibody of rabbit or mouse origin binds to the corresponding target antigen, the biotinylated antibody in this kit • • Rabbit/mouse universal secondary antibody This reagent kit is designed based on the principle that biotin and Streptavidin have a strong affinity. After the primary antibody of rabbit or mouse origin binds to the corresponding target antigen, the biotinylated antibody in this kit • • Rabbit/mouse universal secondary antibody specifically binds to the primary antibody; The biotin labeled on the secondary antibody binds to streptavidin labeled with peroxidase (HRP), forming an antigen-specific primary antibody biotinylated secondary antibody streptavidin complex labeled with HRP. HRP can catalyze substrate colorimetry, thereby inferring the presence and distribution of the tested antigen. The biotinylated secondary antibody and SA-HRP used in this reagent kit all adopt optimized labeling and purification techniques, which make their staining more sensitive and have a lower background. They are suitable for detecting formalin fixed paraffin embedded tissue sections, as well as frozen sections, cell slides, freshly prepared blood smears, etc. The rabbit/mouse universal Streptavidin HRP kit is suitable for use with aladdin ready to use or concentrated antibodies. Composition：Note: This reagent kit is only suitable for IHC experiments where the primary antibody is an immune or mouse derived antibodNotes:1. Add 1 drop (approximately 50) to each slice µ l) Calculation: 3ml can make 60 slices, and 18ml can make 360 slices.2.For tissues with abundant endogenous biotin content, it is best to use endogenous biotin blockers for blocking when using this kit.3. DAB working solution is prepared and used immediately, and the prepared working solution is effective within 1 hour in the dark at 2-8 ° C.4. During the experiment, avoid drying the tissue slices, so the amount of working fluid used during each incubation step must be sufficient to ensure complete coverage of the tissue sample, and incubation should be carried out in a wet box as much as possible.5. To obtain the best experimental results, please make sure to optimize the experimental conditions and reagent dosage.6. DAB is a suspected carcinogen, please take necessary protective measures when using it. 7. This product is only for scientific research and cannot be used for human reactions or treatments.Operation steps：1. Routine processing of samples such as paraffin or frozen tissue sections or cell slides to be tested.1) Preparation for staining of tissue sections or cell slides: a. Dewaxing and hydration of paraffin sections: bake at 60 º C for 1 hour, dewaxing twice with xylene for 5 minutes each time; Then immerse in gradient ethanol (anhydrous ethanol anhydrous ethanol 95% 85% 75% ethanol) and distilled water for 5 minutes each for hydration. b. Frozen sections and cell climbing sections (or climbing sections) were soaked in 0.01 M pH 7.4 PBS and washed 3 times for 5 minutes. Then cover the tissue (or cells) with 0.1% Triton X-100 and infiltrate for 15 minutes. Wash twice with 0.01 M pH 7.4 PBS for 5 minutes.2) Antigen repair of paraffin sections: In most cases, high-pressure repair with citric acid buffer is suitable for paraffin tissue sections. Preparation of repair solution: Add 10 ml of citric acid buffer (IHC antigen repair solution, 100 x) to 1 L of deionized water, and mix well. Repair process: The repair solution is added to a high-pressure cooker, and the repaired slices are immersed in the repair solution (must have no tissue). Cover the pressure cooker cover, heat until evenly sprayed with steam, and start timing from the spraying. After 1-2 minutes, the pressure cooker leaves the heat source and cools naturally to room temperature. Remove the slices, rinse with distilled water, and rinse twice with PBS (0.01 M pH 7.4) for 3 minutes each time.2. Add an appropriate amount of Solution A white solution, which is an endogenous peroxidase blocking solution, and incubate at room temperature for 10 minutes, then rinse thoroughly with PBS.3. Add an appropriate amount of Solution B white solution dropwise, which is sealed with normal sheep serum working solution. Incubate at room temperature for 10 minutes and shake dry.4. Add an appropriate amount of primary antibody working solution (commercial ready to use antibodies or concentrated antibodies diluted in appropriate proportions) dropwise, incubate according to experimental requirements, and then rinse thoroughly with PBS.5. Add an appropriate amount of Solution C yellow solution, namely biotin labeled sheep anti rabbit/mouse secondary antibody working solution, incubate at room temperature for 10 minutes, and rinse thoroughly with PBS.6. Add an appropriate amount of Solution D red solution, which is HRP labeled streptavidin. Incubate at room temperature for 10 minutes and rinse thoroughly with PBS.7. Preparation of DAB color working solution: According to the required amount, mix DAB-A and DAB-B in a volume ratio of 1:19 to obtain DAB color working solution. Alternatively, one drop (approximately 50) can be added per milliliter of reagent B µ l) Reagent A, mix well.8. Color development: Add an appropriate amount of DAB color development working solution to the tissue section or cell slide that needs to be developed, and the color development time is generally 1-5 minutes. Observe and control the color development time under a microscope. When the optimal color development effect is achieved, rinse with tap water to terminate the color development. The colored slices are re stained, dehydrated and transparent, and can be stored for a long time after sealing... Read More | 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 |