| Description | Reconstitution: After centrifugation, we suggest addition of 50 mM Na Acetate, pH 5.5, with 150 mM NaCl to the original volume, followed by gentle mixing to ensure adequate homogenization.This enzyme degrades elastin, collagen, and proteoglycan and has been implicated in the development of pulmonaryReconstitution: After centrifugation, we suggest addition of 50 mM Na Acetate, pH 5.5, with 150 mM NaCl to the original volume, followed by gentle mixing to ensure adequate homogenization.This enzyme degrades elastin, collagen, and proteoglycan and has been implicated in the development of pulmonary emphesema and rheumatoid arthritis.Activity: 20-22 units per mg protein. One unit is defined as the amount of enzyme that hydrolyzes one umole of MeO-Suc-Ala-Ala-Pro-Val-pNA per minute at 25℃, pH 7.5. Product Citations:Mittendorf, Elizabeth A., Gheath Alatrash, Na Qiao, Yun Wu, Pariya Sukhumalchandra, Lisa S. St John, Anne V. Philips et al. "Breast cancer cell uptake of the inflammatory mediator neutrophil elastase triggers an anticancer adaptive immune response." Cancer research 72, no. 13 (2012): 3153-3162van den Berg, Carmen W., et al. "Mechanism of Neutrophil Dysfunction: Neutrophil Serine Proteases Cleave and Inactivate the C5a Receptor." The Journal of Immunology 192.4 (2014): 1787-1795.Edwards, J. Vincent, Nicolette Prevost, Kandan Sethumadhavan, Abul Ullah, and Brian Condon. "Peptide conjugated cellulose nanocrystals with sensitive human neutrophil elastase sensor activity." Cellulose (2013): 1-13.Valdez C, Wong YC, Schwake M, Bu G, Wszolek ZK, Krainc D.Progranulin-mediated deficiency of cathepsin D results in FTD and NCL-like phenotypes in neurons derived from FTD patients.Hum Mol Gen. 2017. 2017 Sep 25. doi:10.1093/hmg/ddx364Kerros C, et al.Neuropilin-1 mediates neutrophil elastase uptake and cross-presentation in breast cancer cells.J Biol Chem. 2017 Jun 16;292(24):10295-10305. doi: 10.1074/jbc.M116.773051. Epub 2017 May 3.Martin KR, Kantari-Mimoun C, Yin M, Pederzoli-Ribeil M, et al.Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles.J Biol Chem. 2016 May 13;291(20):10476-89. doi: 10.1074/jbc.M115.698639. Epub 2016 Mar 9.Edwards JV, Fontenot KR, Prevost NT, Pircher N, Liebner F, Condon BD.Preparation, Characterization and Activity of a Peptide-Cellulosic Aerogel Protease Sensor from Cotton.Sensors (Basel). 2016 Oct 26;16(11). pii: E1789.Chawla A, Alatrash G, Philips AV, Qiao N, et al.Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells.Cancer Immunol Immunother. 2016 Jun;65(6):741-51. doi: 10.1007/s00262-016-1841-6. Epub 2016 Apr 29.Sinden NJ, et al.α-1-Antitrypsin variants and the proteinase/antiproteinase imbalance in chronic obstructive pulmonary disease.Am J Physiol Lung Cell Mol Physiol. 2015 Jan 15;308(2):L179-90.Ksiazek M, et al.Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia, Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop.J Biol Chem. 2015 Jan 2;290(1):658-70. doi: 10.1074/jbc.M114.601716. Epub 2014 Nov 11.Ramadass M, Ghebrehiwet B, Kew RR.Enhanced recognition of plasma proteins in a non-native state by complement C3b. A possible clearance mechanism for damaged proteins in blood.Mol Immunol. 2015 Mar;64(1):55-62. doi: 10.1016/j.molimm.2014.10.022. Epub 2014 Nov 15.Thomas MP, et al.Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins.J Immunol. 2014 Jun 1;192(11):5390-7. doi: 10.4049/jimmunol.1303296. Epub 2014 Apr 25.Ungurs MJ, Sinden NJ, Stockley RA.Progranulin is a substrate for neutrophil-elastase and proteinase-3 in the airway and its concentration correlates with mediators of airway inflammation in COPDAm J Physiol Lung Cell Mol Physiol. 2014 Jan 1;306(1):L80-7. doi: 10.1152/ajplung.00221.2013. Epub 2013 Nov 1.Sinden NJ, Koura F, Stockley RA.The significance of the F variant of alpha-1-antitrypsin and unique case report of a PiFF homozygote.BMC Pulm Med. 2014 Aug 7;14:132. doi: 10.1186/1471-2466-14-132.Kenninston JA, et al.Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody.J Biol Chem. 2014 Aug 22;289(34):23596-608. doi: 10.1074/jbc.M114.569061. Epub 2014 Jun 26.Tejera P, et al.Functional Characterization of Polymorphisms in the Peptidase Inhibitor 3 (Elafin) Gene and Validation of Their Contribution to Risk of Acute Respiratory Distress Syndrome.Am J Respir Cell Mol Biol. 2014 Aug;51(2):262-72. doi: 10.1165/rcmb.2013-0238OC.Zheng K, et al.Influence of glycosylation pattern on the molecular properties of monoclonal antibodies.MAbs. 2014 May-Jun;6(3):649-58. doi: 10.4161/mabs.28588. Epub 2014 Mar 24... Read More | Cardiolipin is a unique phospholipid present in the inner mitochondrial membrane, which makes up to 20% of total lipids. It is a non-bilayer anionic phospholipid, which has four acyl chains and small headgroupHeart CA has been used as a standard stock solution for its quantitative analysis using Cardiolipin is a unique phospholipid present in the inner mitochondrial membrane, which makes up to 20% of total lipids. It is a non-bilayer anionic phospholipid, which has four acyl chains and small headgroupHeart CA has been used as a standard stock solution for its quantitative analysis using liquid chromatography?mass spectrometry (LC-MS)/MS. It has also been used for liposome preparation... Read More | Collagenase NB 1 is chromatographically highly purified; therefore it contains a very high collagenolytic activity. It is largely free from additional enzymatic activities like clostripain, trypsin-like activity and neutral protease, as well as endotoxins.SpecificationsContains chromatographically Collagenase NB 1 is chromatographically highly purified; therefore it contains a very high collagenolytic activity. It is largely free from additional enzymatic activities like clostripain, trypsin-like activity and neutral protease, as well as endotoxins.SpecificationsContains chromatographically highly purified class I and class II collagenase (1).Largely free from clostripain, trypsin-like protease and neutral protease.Vial contains not less than 2000 PZ U collagenase.Activity (Wünsch): ≥ 3.00 U/mgEndotoxin: ≤ 10.0 EU/mg (Ph. Eur.)(Clostridiopeptidase A)EC 3.4.24.3 • Mr ca. 70 000 - 120 000 (collagenases) • CAS [9001-12-1]ApplicationCollagenase NB 1 is, mostly in combination with Neutral Protease NB, suitable for cell isolation from several tissue types.References and DefinitionsUnit definition: Collagenase: 1 U according to Wünsch (2) catalyzes the hydrolysis of 1 µmole 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucylglycyl-L-prolyl-D-arginine per minute at 25 °C, pH 7.1.Endotoxin: Ph. Eur. (1 Endotoxin Unit is equal to 1 International Unit of a WHO approved reference standard endotoxin (RSE)).References1. Bond, M.D. & van Wart, H.E. (1984) Biochemistry 23, 3077-30912. Wünsch, E. & Heidrich, H.G. (1963) Hoppe-Seyler's Z. Physiol. Chem. 333, 149-51... 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>97% SDS-PAGE.FunctionReceptor for interleukin-2 |