| 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 | Inquire | Inquire | Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：Histones are a complex family of highly conserved basic proteins responsible for packaging chromosomal DNA into nucleosomes. Histone proteins exhibit two levels of diversity: 1. evolutionary diversity Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：Histones are a complex family of highly conserved basic proteins responsible for packaging chromosomal DNA into nucleosomes. Histone proteins exhibit two levels of diversity: 1. evolutionary diversity between species and 2. subtype diversity in a class(H1, H2A, H2B, H3 or H4) within a species. It has become more and more evident that histone modifications are key players in the regulation of chromatin states and dynamics as well as in gene expression. Therefore, histone modifications and the enzymatic machinery that set them are crucial regulators that can control cellular proliferation, differentiation, plasticity, and malignancy processes. However, extracellular histones are a double-edged sword because they also damage host tissue and may cause death. Histones bound to platelets, induced calcium influx, and recruited plasma adhesion proteins such as fibrinogen to induce platelet aggregation. Histone H2B proteins have been studied in a variety of species and are easily detected in most species. The reversible ubiquitylation of histone H2B has long been implicated in transcriptional activation and gene silencing. Phosphorylation of H2B serine 32 occurs in normal cycling and mitogen-stimulated cells. Notably, this phosphorylation is elevated in skin cancer cell lines and tissues compared with normal counterparts. HIST2H2BE is a member of the histone H2B family and generates two transcripts through the use of the conserved stem-loop termination motif, and the polyA addition motif... Read More | Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description: High-mobility group box 1 protein (HMGB1), also known as HMG-1 or amphoterin previously, is a member of the HMGB family consisting of three members, HMGB1, HMGB2, and HMGB3. HMGB1 is a DNA-binding nuclear protein,Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description: High-mobility group box 1 protein (HMGB1), also known as HMG-1 or amphoterin previously, is a member of the HMGB family consisting of three members, HMGB1, HMGB2, and HMGB3. HMGB1 is a DNA-binding nuclear protein, released actively following cytokine stimulation as well as passively during cell death. It is the prototypic damage-associated molecular pattern (DAMP) molecule and has been implicated in several inflammatory disorders. HMGB1 signals via the receptor for advanced glycation end-product (RAGE) and members of the toll-like receptor (TLR) family. The most prominent HMGB1 protein and mRNA expression arthritis are present in pannus regions, where synovial tissue invades articular cartilage and bone. HMGB1 promotes the activity of proteolytic enzymes, and osteoclasts need HMGB1 for functional maturation. As a non-histone nuclear protein, HMGB1 has a dual function. Inside the cell, HMGB1 binds DNA, regulating transcription, and determining chromosomal architecture. Outside the cell, HMGB1 can serve as an alarmin to activate the innate system and mediate a wide range of physiological and pathological responses. Extracellular HMGB1 represents an optimal " necrotic marker" selected by the innate immune system to recognize tissue damage and initiate reparative responses. However, extracellular HMGB1 also acts as a potent pro-inflammatory cytokine that contributes to the pathogenesis of diverse inflammatory and infectious disorders. HMGB1 has been successfully therapeutically targeted in multiple preclinical models of infectious and sterile diseases including arthritis. As shown in studies on patients as well as animal models, HMGB1 can play an important role in the pathogenesis of the rheumatic disease, including rheumatoid arthritis, systemic lupus erythematosus, and polymyositis among others. Besides, enhanced postmyocardial infarction remodeling in type 1 diabetes mellitus was partially mediated by HMGB1 activation... Read More |