| Description | Aladdin's E. coli DNA Polymerase I purified using the PerfectProtein™ technology platform developed by aladdin, catalyzes the the 5'→3' polymerization of deoxyribonucleotides in a DNA template-dependent manner [1]. It also possesses dsDNA nick-specific 5'→3' exonuclease activity, Aladdin's E. coli DNA Polymerase I purified using the PerfectProtein™ technology platform developed by aladdin, catalyzes the the 5'→3' polymerization of deoxyribonucleotides in a DNA template-dependent manner [1]. It also possesses dsDNA nick-specific 5'→3' exonuclease activity, ssDNA-specific 3'→5' exonuclease activity, and RNase H activity [2]. The DNA synthesis activity and dsDNA nick-specific 5'→3' exonuclease activity allow for DNA synthesis starting from a 3'-OH group at the nick and degradation of single-stranded DNA at 5' end, facilitating gap filling. Its ssDNA-specific 3'→5' exonuclease activity serves a proofreading function during DNA synthesis. In the presence of dNTP, the E. coli DNA Polymerase I primarily exhibits DNA polymerase activity, while in the absence of dNTP, it displays more ssDNA-specific exonuclease activity, such as 5'→3' exonuclease activity on either strand at the 5' end of a blunt-ended dsDNA.The versatility of E. coli DNA Polymerase I allows it to initiate the synthesis of new DNA chains from gaps or nicks in dsDNA, and to degrade the DNA strand complementary to the template strand from the nick, enabling nick translation. It also ensures proofreading of mismatch during DNA replication and fills in gaps that occur during replication and repair processes [3].Please refer to Figure 1 for the performance of this product in filling 5' overhangs of dsDNA.Figure 1. Performance of Aladdin's E. coli DNA Polymerase I in filling 5' overhangs of dsDNA. In a 20µl reaction (10mM Tris-HCl, 50mM NaCl, 10mM MgCl2, 1mM DTT, 100µM dNTP Mix, 0.5µM dsDNA with 5' overhang, pH 7.9 at 25℃), the specified amount of this product or E. coli DNA Polymerase I from Company N (Competitor) was added. After incubation at 37℃ for 20 minutes, reactions were terminated by incubation at 75℃ for 20 minutes, followed by 15% native PAGE analysis of 5µl of the reaction product after mixing with 1µl of 6X DNA Loading Buffer. Electrophoresis was conducted at 180V for 60 minutes, and then the gel was stained with Gel-Red (10000X) at room temperature for 5 minutes. The experimental results were observed under a UV lamp. As shown in the figure, this product has similar catalytic efficiency to Competitor. The substrate dsDNA with 5' overhang was obtained by annealing 5'-ATACATAGATACATAGACTGGCCGTCGTTTTAC-3' and 5'-GTAAAACGACGGCCAGT-3' using Annealing Buffer for DNA Oligos (5X) according to manufacture's instructions. This figure is for reference only, which may vary due to different experimental conditions.Please refer to Figure 2 for performance of this product in digesting double-stranded linear DNA with amino-modified 3' ends.Figure 2. Performance of Aladdin's E. coli DNA Polymerase I in digesting amino-modified 3' ends of dsDNA (5'→3' exonuclease activity). In a 20µl reaction (10mM Tris-HCl, 50mM NaCl, 10 mM MgCl2, 1mM DTT, 0.5µM dsDNA), the specified amount of this product or E. coli DNA Polymerase I from Company N (Competitor) was added. After incubation at 37℃ for 20 minutes, reactions were terminated by incubation at 75℃ for 20 minutes, followed by 15% native PAGE analysis of 5µl of the reaction product after mixing with 1µl of 6X DNA Loading Buffer. Electrophoresis was conducted at 180V for 60 minutes, and then the gel was stained with Gel-Red (10000X) at room temperature for 5 minutes. The experimental results were observed under a UV lamp. As shown in the figure, this product has similar catalytic efficiency to Competitor. The substrate dsDNA with amino-modified 3' ends was obtained by annealing 5'-ATACATAGATACATAGACTGGCCGTCGTTTTAC-3'NH2 and 5'-GTAAAACGACGGCCAGTCTATGTATCTATGTAT-3'NH2 using Annealing Buffer for DNA Oligos (5X) according to manufacture's instructions. This figure is for reference only, which may vary due to different experimental conditions.sApplication：DNA synthesis; complementary filling of dsDNA 5' overhangs; removal of dsDNA 3' overhangs; second strand cDNA synthesis [4]; in combination with DNase I for DNA nick translation; nick translation to obtain probes with high specific activity.Source：Purified from E. coli with recombinant expression of E. coli DNA Polymerase I.Enzyme storage buffer：25mM Tris-HCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol (pH 7.4 at 25 ℃).Inactivation or inhibition：This product can be inactivated by incubation at 75℃ for 20 minutes.Precautions：Due to the exonuclease activity of E. coli DNA Polymerase I, please avoid high environmental temperatures before performing the reaction. Otherwise, the DNA strands may be cleaved.E. coli DNA Polymerase I does not possess endonuclease activity, nor DNase I either. Therefore, when performing nick translation reactions, DNase I must be added.Vigorous shaking or stirring of E. coli DNA Polymerase I can cause enzyme inactivation.E. coli DNA Polymerase I has a high affinity for DNA. Addition of excessive amount of enzyme may lead to aggregation, thus affecting the amplification reactions.E. coli DNA Polymerase I can polymerize deoxyribonucleotides labeled with biotin, digoxigenin, or fluorescence, etc, allowing for synthesis of labeled DNA probes.The enzyme should be kept on ice during use, and stored at -20℃ immediately after use.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1. Fill-in of 5' overhangs of dsDNAa. Set up the following reaction on ice.ReagentVolumeFinal ConcentrationNuclease-free Water(16-x)µl-dsDNA with 5' overhangsxµl~0.5µM or 5-200ng/µl10X Reaction Buffer2µl1XdNTP Mix (2mM each)1µl100µME.coli DNA Polymerase I (10U/µl)1µl0.5U/µlTotal Volume20µl-Note 1: The enzyme amount can be reduced appropriately to avoid template cleavage due to its exonuclease activity.Note 2: When multiple reactions are required, prepare a master mix including all reagents except for dsDNA, and then dispense to different nuclease-free PCR tubes. Finally, add dsDNA template to each tube.Note 3: If the dsDNA with 5' overhangs are oligonucleotides, the final concentration can be approximately 0.5µM. However, for digested DNA plasmids, the final concentration can be approximately 5-200ng/µl.b. Mix well gently and then have a pulse-spin in a microfuge to collect the liquid at the bottom of the tube.c. Incubate at 37℃ for 20 minutes. Note: The reaction time can be adjusted based on actual situations.d. Incubate at 75℃ for 20 minutes to inactivate the E. coli DNA Polymerase I.2. Digestion of Double-stranded Linear DNAa. Set up the following reaction on ice.ReagentVolumeFinal ConcentrationNuclease-free Water(17-x)µl-dsDNAxµl~0.5µM or 5-200ng/µl10X Reaction Buffer 2µl1XE.coli DNA Polymerase I (10U/µl)1µl0.5U/µlTotal Volume20µl-Note: When multiple reactions are required, prepare a master mix including all reagents except for dsDNA, and then dispense to different nuclease-free PCR tubes. Finally, add dsDNA to each reaction tube.b. Mix well gently and then have a pulse-spin in a microfuge to collect the liquid at the bottom of the tube.c. Incubate at 37℃ for 20 minutes. Note: The reaction time can be adjusted based on actual situations.d. Incubate at 75℃ for 20 minutes to inactivate E. coli DNA Polymerase I.3. For other applications, please refer to appropriate literature.FAQ:1. Can the E. coli DNA Polymerase I fill in 3' overhangs?No, E. coli DNA Polymerase I cannot fill in 3' overhangs. It can only generate blunt ends by removing 3' overhangs. 's E. coli DNA Polymerase I, Klenow Fragment, and T4 DNA Polymerase can be used for fill-in of 3' overhangs.2. Can E. coli DNA Polymerase I fill in 5' overhangs of DNA?Yes, E. coli DNA Polymerase I can fill in 5' overhangs of dsDNA. Klenow Fragment (, D7037) lacks 5'→3' exonuclease activity and is recommended for fill-in of 5' overhangs.3. Can E. coli DNA Polymerase I be used for nick translation experiments?Yes, nick translation experiments are one of the important applications of E. coli DNA Polymerase I.4. Are there temperature requirements for nick translation experiments?The incubation temperature for nick translation experiments should be below 20℃. At higher temperatures, the newly synthesized DNA can separate and be replicated.5. Can E. coli DNA Polymerase I be heat-inactivated?Yes, this product can be inactivated by heating at 75℃ for 20 minutes. Addition of 10mM EDTA to chelate Mg2+ before performing heat-inactivation can protect the DNA ends. 6. Can E. coli DNA Polymerase I remove 5' overhangs?No, the 5'→3' exonuclease activity of this product is only applicable to gaps in dsDNA.7. Can DNA nick translation be used for labeling probes?Yes, this product can remove template bases at nicks using its 5'→3' exonuclease activity and fill in nicks with labeled nucleotides. This method is suitable for generating large and uniform probes, but with lower efficiency probably.References:1. Kunkel TA, Loeb LA, Goodman MF. J Biol Chem. 1984. 259(3):1539-45.2. Green MR, Sambrook J. Cold Spring Harb Protoc. 2020. 2020(5):100743.3. Yu H, Chao J, Patek D, Mujumdar R, Mujumdar S, Waggoner AS. Nucleic Acids Res. 1994. 22(15):3226-32.4. D'Alessio JM, Gerard GF.Nucleic Acids Res. 1988. 16(5):1999-2014... Read More | Protein Purity≥85% by SDS PAGEExtinction CoeffA280 nm = 0.974 at 1.0 mg/ml for pure C3bMolecular Weight185,000 Da (2 chains)General DescriptionCynomolgus monkey C3 (cyno C3) is purified from pooled normal cynomolgus monkey serum. C3 is central to the activation of all three pathways of Protein Purity≥85% by SDS PAGEExtinction CoeffA280 nm = 0.974 at 1.0 mg/ml for pure C3bMolecular Weight185,000 Da (2 chains)General DescriptionCynomolgus monkey C3 (cyno C3) is purified from pooled normal cynomolgus monkey serum. C3 is central to the activation of all three pathways of complement activation (Law, S.K.A. and Reid, K.B.M. (1995)). Initiation of each pathway generates proteolytic enzyme complexes (C3 convertases) which are bound to the target surface. These enzymes cleave a peptide bond in C3 releasing the anaphylatoxin C3a and activating C3b. For a brief time (~60 µs) this nascent C3b is capable of reacting with and covalently coupling to hydroxyl groups on the target surface. Carbohydrates are the favored target, but protein hydroxyls and amino groups also react. This process of tagging the target surface with C3b is called opsonization. The reactive site in nascent C3b is a thioester (Tack B.J., et al. (1980); Pangburn M.K. and MüllerEberhard H.J. (1980)) and C3b is linked to the target through a covalent ester bond (an amide bond is formed if C3b is attached to amino groups). Most of the C3 activated during complement activation never attaches to the surface because its thioester reacts with water forming fluid phase C3b which is rapidly inactivated by factors H and I forming iC3b. Surface-bound C3b is necessary in all three pathways for efficient activation of C5 and formation of C5b-9 complexes that lyse the target cell membrane. Surface-bound C3b and its breakdown products iC3b and C3d are recognized by numerous receptors on lymphoid and phagocytic cells which use the C3b ligand to stimulate antigen presentation to cells of the adaptive immune system. The end result is an expansion of target-specific B-cell and T-cell populations.Physical Characteristics & StructureCynomolgus monkey C3 is an uncharacterized protein. The calculated molecular weight based on its amino acid sequence is 184,926 daltons similar to that of human C3 (185,000 daltons). Like human C3, cyno C3 is composed of two disulfide-linked chains. Analysis of purified cyno C3 by SDS/polyacrylamide gel electrophoresis under non-reduced conditions shows the mobility of cyno C3 to be similar to that of human C3. Under reduced conditions, the migration of the alpha chain of cyno C3 is comparable to that of human C3 alpha chain (110,000 daltons) while the beta chain migrates slightly ahead of the human C3 beta chain (75,000daltons).The extinction coefficient of cyno C3 is calculated from its amino acid sequence using ProtParam and assumes all pairs of Cys residues form cystines (i.e. a pair of cystine molecules are joined by a disulfide bond). The theoretical pI value for cyno monkey C3 is 6.03. Employing immunoturbidimetric method the serum concentration of cyno C3 has been reported to be 1.27 mg/ml in males and 1.1 mg/ml in female monkeys (Park H-K et al., (2016)). FunctionThe biological functions of C3 are described above in the General Description and Physical Characteristics sections.GeneticsCynomolgus monkey C3 chromosome location 19. The NCBI Gene ID number for Cynomolgus monkey C3 is 102131458 and UniProt accession number is A0A2K5VPN1.Precautions/Toxicity/HazardsThis protein is purified from animal serum and therefore precautions appropriate for handling any animal blood-derived product must be used.ReferencesLaw, S.K.A. and Reid, K.B.M. (1995) Complement 2nd Edition (ISBN 0199633568) Oxford University Press, Oxford.Tack BF, Harrison RA, Janatova J, Thomas ML, Prahl JW. (1980) Evidence for presence of an internal thiolester bond in third component of human complement. Proc Natl Acad Sci U S A. 77:5764-8.Pangburn M.K. and Müller-Eberhard H.J. (1980) Relation of putative thioester bond in C3 to activation of the alternative pathway and the binding of C3b to biological targets of complement. J Exp Med. 152:1102-14.Park H-K, Cho J-W, Lee B-S, Park H, Han J-S, Yang M-J, Im W-J, Park D-Y, Kim W-J, Han SC, Kim Y-B. (2016) Reference values of clinical pathology parameters in cynomolgus monkeys (Macaca fascicularis) used in preclinical studies. Lab Anim Res. 32(2):79-86... Read More | Inquire | Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description: DCX (doublecortin, N-GST chimera)contains 2 doublecortin domains and belongs to the doublecortin family. It is highly expressed in neuronal cells of fetal brain, but not expressed in other fetal tissues. In the Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description: DCX (doublecortin, N-GST chimera)contains 2 doublecortin domains and belongs to the doublecortin family. It is highly expressed in neuronal cells of fetal brain, but not expressed in other fetal tissues. In the adult, it is highly expressed in the brain frontal lobe, but very low expression in other regions of brain, and not detected in heart, placenta, lung, liver, skeletal muscles, kidney and pancreas. DCX is a microtubule-associated protein required for initial steps of neuronal dispersion and cortex lamination during cerebral cortex development. It may act by competing with the putative neuronal protein kinase DCAMKL1 in binding to a target protein. DCX may in that way participate in a signaling pathway that is crucial for neuronal interaction before and during migration, possibly as part of a calcium ion-dependent signal transduction pathway. It may be part with LIS-1 of a overlapping, but distinct, signaling pathways that promote neuronal migration. Defects in DCX are the cause of lissencephaly X-linked type 1 and subcortical band heterotopia X-linked... Read More | Purity: >90%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description: Major histocompatibility complex, class II, DR alpha (HLA-DRA) belongs to the MHC class II family. HLA-DRA binds peptides derived from antigens which access the endocytic route of antigen presenting cells (APC) Purity: >90%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description: Major histocompatibility complex, class II, DR alpha (HLA-DRA) belongs to the MHC class II family. HLA-DRA binds peptides derived from antigens which access the endocytic route of antigen presenting cells (APC) and presents them on the cell surface for identification by the CD4 T-cells. The peptide binding cleft accommodates peptides of 10-30 residues. The peptides presented by MHC class II molecules are generated mainly by degradation of proteins which access the endocytic route, where they are processed by lysosomal proteases and other hydrolases... Read More |