Deoxyribonuclease I (DNase I), from bovine pancreas HYC118
DNase I is an endonuclease that can digest single-stranded and double-stranded DNA to produce single deoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides.It can hydrolyze the phosphodiester bond to produce monodeoxynucleotides and oligodeoxynucleotides containing 5'-phosphate groups and 3'-OH groups. The average digestion product is the smallest polytetranucleotide. DNase I can catalyze many forms of DNA, such as single-stranded DNA,double-stranded DNA, and even chromatin(its cutting rate is affected by histones).The optimum pH range is 7-8. The activity of DNase I depends on Ca²+ and can be activated by divalent metal ions,such as Co2+, Mn²+, Zn²+, etc. 5mM Ca²+ can protect the enzyme from being hydrolyzed. In the presence of Mg²+, the enzyme can recognize and cut any site on any strand of DNA randomly;and in the presence of Mn²+, it can recognize two strands of DNA at the same time and cut at almost the same site to form blunt ends, or sticky ends with 1-2 nucleotides protruding. DNase I was first isolated from pancreas, and mammalian pancreas is still a major source of the enzyme. This product is derived from bovine pancreas and consists of four chromatographically distinct components A, B, C and D in a molar ratio of 4:1:1, with component D being very small. It is often used in molecular biology experiments to remove DNA from proteins or to create gaps in DNA to allow the insertion of labelled bases into DNA. This product is supplied as a lyophilised powder containing calcium chloride with an enzyme activity of ≥2000 Kunitz units/mg protein.
Components
|
Components |
Size-1 |
Size-2 |
|
Deoxyribonuclease I (DNase I), from bovine pancreas |
15 KU |
10 × 15 KU |
Storage
Store at-15°C ~-25℃ for 2 year.
Unit Definition
Under the conditions of 25°C and pH 5.0, DNase catalyses the DNA, and the change in ΔA260 per ml per minute increase of 0.001 is defined as one enzyme activity unit (Kunitz unit).
Specification
|
CAS No. |
9003-98-9 |
|
Molecular Weight |
~31 kDa |
|
Kunitz Units |
≥2000 Kunitz Units/mg protein |
|
Type |
Type IV |
|
Optimal pH |
7～8 |
|
Appearance |
White to light brown freeze-dried powder |
|
Purity |
Protein: ≥80% by Biuret |
|
Solubility |
0.15 M NaCl: 5 mg/mL, colorless and transparent solution |
|
Activators |
Various divalent metal ions such as Mg2+、Mn2+, Ca2+, Co2+, and Zn2+ |
|
Inhibitors |
β-Mercaptoethanol; Chelating agent; SDS; Actin |
|
Storage Solution |
20 mM sodium acetate (pH 6.5), 5 mM CaCl2, 0.1 mM PMSF, 50% glycerol. |
DNase I inactivation or inhibition
After addition of EDTA to a final concentration of 2.5mM, heating at 65°C for 10 min can inactivateDNaseI. Phenol-chloroform extraction can also inactivate DNase I. Metal ion chelators, zinc ions at millimolar/litre concentrations, 0.1% SDS, reducing agents such as DTT and mercaptoethanol, and salt concentrationsabove50-100 mM all have significant inhibitory effects on DNase I.
Instructions
Applied to protein extraction experiments, for reference only
1. Reaction system: Add DNase I storage solution at 1/100 volume to the protein extract (to giveafinal concentration of 20 U/mL) and add 1 M MgCl2 at 1/100 volume.
2. Reaction conditions: 37℃, 30-60 min. Continue with the following protein extraction experiments.
Note: Since EDTA can chelate Ca2+ and Mg2+ required for enzyme activity, EDTA must be removed from the initial protein lysis solution, otherwise it will reduce the digestive capacity of DNase I.