| 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 | Biochemical Test:SDS-PAGE (purity> 80%); Western blot with patient sample.Calculated Isoelectric Point:pH 8.83 | Product DescriptionEndo F2 cleaves N-linked (asparagine-linked) biantennary oligosaccharides from glycoproteins. It also will cleave high mannose glycans but at a 40x reduced rate. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, Product DescriptionEndo F2 cleaves N-linked (asparagine-linked) biantennary oligosaccharides from glycoproteins. It also will cleave high mannose glycans but at a 40x reduced rate. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact.Endoglycosidase F2 is less sensitive to protein conformation than PNGase F and is therefore more suitable for deglycosylation of native proteins. However, for optimal results, denaturation of the glycoprotein is recommended.Contents60 µl aliquot of enzyme (0.3 U) in 10 mM sodium acetate 25mM NaCl, pH 4.5Included with 20 µL and 60 µL pack sizes:5x Reaction Buffer – 250 mM sodium acetate, pH 4.5Molecular weight 32,000 daltonsSpecific Activity Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured porcine fibrinogen in 1 minute at 37°C, pH 5.5. Cleavage is monitored by SDS-PAGE (cleaved fibrinogen migrates faster).Formulation The enzyme is provided as a sterile-filtered solution in 10 mM sodium acetate, 25mM NaCl, pH 4.5Specificity Endo F2 cleaves Asparagine-linked biantennary and high mannose glycans (at a 40X reduced rate). It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Endoglycosidase F2 is less sensitive to protein conformation than PNGase F and is therefore more suitable for deglycosylation of native proteins. However for optimal results, denaturation of the glycoprotein is recommended.Quality & Purity Endo F2 is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases.Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly.Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 38 µl final volume with de-ionized water. 2. Add 10 µl 5x Reaction Buffer 4.5 3. Add 2.0 µl of Endo F2 to the reaction. Incubate 1 hour at 37°C. Monitor cleavage by SDS-PAGEThe production host strain has been extensively tested and does not produce any detectable glycosidases... Read More | Products contentN665730Component24 T96 TStorageN665730ATPS V50 144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730B5×FA Reaction Buffer144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730C2×HiFidelity PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw Products contentN665730Component24 T96 TStorageN665730ATPS V50 144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730B5×FA Reaction Buffer144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730C2×HiFidelity PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw cycle.N665730DPPM48 µL192 µL-20℃. Avoid freeze/thaw cycle.* This kit is suitable for human genomic DNA library construction with a starting template DNA input of 50 ng. We also have transposase library construction kits for human genomic DNA starting at 5 ng and 1 ng, so it is recommended to use different kits for different starting amounts of DNA in order to obtain higher quality libraries. Products IntroductionThis kit is developed for Illumina's high-throughput sequencing platform and provides the enzyme premix system and reaction buffer for genomic DNA library construction, including all components except PCR primers. Compared with the traditional library construction kits, this kit adopts the new transposase method for library construction, which can complete DNA fragmentation, end repair and junction reaction in one simple enzymatic reaction, significantly reducing the amount of template, reducing the number of experimental steps, and shortening the time of library construction; it adopts the high-fidelity DNA polymerase for library enrichment, and the preference-free PCR amplification can expand the coverage area of the sequence, which can be used for efficient and effective sequencing. The use of high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification broadens the coverage area of the sequence and enables efficient preparation of DNA libraries for Illumina's second-generation sequencing platform. The kit is suitable for DNA libraries with a starting template of 50 ng, and all reagents in the kit have been subjected to strict quality control and functional validation to maximize the stability and reproducibility of library construction. Product Features ● DNA fragmentation and junction ligation in one step.● Ultra-fidelity amplification minimizes amplification-preferred steps.Provide your own instruments, kits and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use DNA purification and recovery kit by magnetic bead method.3. Library PCR primer kits: transposase method for second-generation sequencing multi-sample primer kits are recommended. 4. Anhydrous ethanol, deionized water (pH between 7.0 and 8.0).5. Reaction tubes: It is recommended to use low adsorption PCR tubes and 1.5 ml centrifuge tubes. Tips: It is recommended to use high quality filter tips to prevent contamination of kits and library samples. Pre-experiment Preparation and Important Notes1. Avoid repeated freezing and thawing of reagents.2. PCR products are easily contaminated due to improper operation, resulting in inaccurate results. It is recommended to isolate the PCR reaction system preparation area from the PCR product purification area, and to use special pipettes to clean the experimental areas at regular intervals.3. Bead purification: the beads should be equilibrated to room temperature before use, all operations on the beads should be carried out at room temperature, 80% ethanol should be dispensed freshly, the beads should be rinsed and dried until the surface is free of liquid reflections and has a frosted appearance, insufficient drying of the beads will cause ethanol residue that will affect the subsequent experiments, and over-drying of the beads will affect the efficiency of DNA recovery.4. The kit is suitable for human genomic DNA library construction, if the DNA sample is a PCR product, it should be ensured that its length>.500 bp, since transposases do not work on DNA ends, it is recommended to extend the PCR product by 50-100 bp at each end of the PCR product to avoid low coverage of the ends for sequencing.Sample PreparationDNA purity requirements: A260/A280 = 1.8-2.0. Sample DNA: dissolve in ultrapure water. DNA Quantification: Too much or too little DNA will affect the quality of the library. It is recommended to use Nano to test the purity of the genomic DNA and then use Qubit to test the concentration of the genome (do not use any absorbance-based assay for template quantification).Schematic diagram of DNA banking processprocedureDNA fragmentation, junction reaction1. Add the following reagents to a 200 µl PCR tube: 2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:DNA should be purified immediately after the fragmentation reaction has been performed and the transposase is still in a high state of activity.to prevent smaller library fragments due to DNA over-fragmentation. Purification of fragmentation productsWe recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Add 50 µl of magnetic beads equilibrated to room temperature to the fragmentation product, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, then add 23 µlddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer 21 µl of supernatant to a new 200 µl PCR tube.PCR amplification Add the following reagents to the 200 µl PCR tube: Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:Selective recovery of library DNA fragmentsIt is recommended to use CombiVision Magnetic Beads DNA Purification and Recovery Kit for selective recovery of DNA fragments. When different sizes of DNA fragments are required, the amount of magnetic beads to be used is different, please refer to the attached table for the specific amount of magnetic beads to be used (if other brands of magnetic beads are used, you need to find out the optimal amount of magnetic beads to be used on your own).Note: Amplification products can also be fragment length sorted and purified using the Gum Recovery Kit. If there is no special requirement for library length distribution, the amplification products can also be purified without selective recovery of DNA fragments as described on page 6 of the manual.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Transfer the PCR product to a 1.5 ml centrifuge tube, rehydrate to 100 µl and add several volumes of magnetic beads equilibrated to room temperature, vortex for 5 seconds and let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, and carefully aspirate the supernatant and transfer it to a new 1.5 ml centrifuge tube.Note: Do not discard the top clear.4. Add several volumes of magnetic beads to the supernatant, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.5. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA.Note: Do not discard the beads.6. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.7. Repeat step 6 once.8. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 20 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.9. Remove the centrifuge tube from the magnetic rack, vortex and oscillate to completely resuspend the beads, and let stand at room temperature for 5 minutes. Leave brieflycentrifuge, place the tube on a magnetic rack until the solution is clear, and transfer the supernatant solution to a new centrifuge tube. Table: Suggested amount of magnetic beads for different segment selection recoveryLibrary DNA fragment purificationWe recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. 50 µl of magnetic beads equilibrated to room temperature were added to the PCR product, vortexed and shaken for 5 seconds, and then left to stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 25 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube.Library quality controlDetermination of library concentrationIn order to obtain high-quality sequencing results, accurate quantification of DNA libraries is required, and the first recommendation is to use Real-timePCR methods are used for absolute quantification of DNA libraries. Additionally, fluorescent dye methods such as the Qubit method or the fluorescent dye picogreen method can be used; do not use quantification methods based on absorbance measurements here. The following approximate formula can be used to convert the molar concentration of the DNA library. Average total length of librariesApproximate conversion formula Library fragment distributionThe prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.Range of segment length distributions... Read More | Purity:>98%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：Heme oxygenase (HMOX) is the rate limiting enzyme in heme catabolism. It cleaves heme to biliverdin, carbon monoxide, and iron. The biliverdin is subsequently converted to bilirubin by biliverdin reductase. Purity:>98%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：Heme oxygenase (HMOX) is the rate limiting enzyme in heme catabolism. It cleaves heme to biliverdin, carbon monoxide, and iron. The biliverdin is subsequently converted to bilirubin by biliverdin reductase. The mechanism of HMOX is unique in that heme serves as the substrate of the enzyme and as the prosthetic group for the activation of iron-bound O2. HMOX activity is highest in spleen where senescent erythrocytes are sequestered and destroyed. Two isoforms, HMOX1 and HMOX2, are expressed in most tissues. HMOX1 is an inducible enzyme in response to heme, heavy metals, oxidative stress, cytokines, and many drugs. Whereas HMOX2 displays a constitutive expression. HMOX1 is expressed mainly in spleen, liver, and kidney, and HMOX2 is prominently expressed in the brain and testes. The increased expression of HMOX1 levels is related to a variety of pathological states, where it functions as a cytoprotective molecule through its by products. HMOX1 also plays important roles in the regulation of cell proliferation, differentiation, and apoptosis... Read More |