The effect of anticancer drugs on DNA topoisomerase activity assay was performed using agarose gel electrophoresis to determine the effect of anticancer drugs on type I and II topoisomerases. Many anticancer drugs target DNA topoisomerases to cause DNA damage by forming breakable DNA-protein complexes.
Operation method
experimental method
Principle
DNA topoisomerases play an important role in the formation of ring and superhelical structures in DNA. On the other hand, the relaxation of DNA superhelical structure, the breakage of single and double strands of DNA and the process of reconnection all depend on the participation of this enzyme, which makes the replication or recombination of DNA possible. Regarding the anticancer drugs, such as hydrocarbons and non-hydrocarbons, many of them are targeting the DNA topoisomerase to make it form the DNA-protein complexes that can be broken and thus cause DNA damage, which is a new research field that has developed in recent years. This is a new area of research that has developed in recent years.
Materials and Instruments
DNA Move I. Preparation of materials For more product details, please visit Aladdin Scientific website.
Dimethyl Sulfoxide Hepes KCl EDTA MgCl2 ATP BSA P4DNA Polyethylene Glycol NaCl
Electrophoresis Absorbance meter Constant temperature water bath Constant temperature incubator Spectrophotometer Centrifuge
1. Drug preparation
(1) Dissolve the drug in 0. mol/l Hepes buffer or in dimethyl sulfoxide containing 10% to 20% or other co-solvents.
(2) The other 80%~90% was 0.1 mol/l Hepes buffer, pH 6.7, so that the final concentration of the drug was 0.2~0.4 mmol/l.
2. Reaction solution
(1) The volume of reaction solution was 24 ul
(2) which contains 500 mmol/l Hepes pH 6.7, 50 mmol/l KCl 100 mmol/l NaCl, 0.1 mmol/l EDTA, 10 mmol/l MgCl2, 0.1 mmol/l ATP, 50 ug/ml BSA, and 0.26ug P4DNA.
II. Experimental steps
1. The experiment grouped the microtubes as
(1) dosed tubes, undosed control tubes, known drug (VP-16) control tubes and standard tubes (containing different concentrations of enzyme, the enzyme dilution of the first tube was determined according to the activity of the enzyme used, and then decreased exponentially to the fourth tube in order, and the fifth tube was a blank tube without enzyme).
(2) Add the enzyme solution (at the same concentration as the first tube of the standard tube) to all tubes except the blank control tube.
(3) Put the reaction in a constant temperature water bath for 30 min and stop the reaction immediately with the termination solution (termination solution: 2% sodium dodecyl, 20% glycerol, 0.05% bromophenol blue).
2. The samples from the above tubes were added to a 1% agarose gel for electrophoresis in electrophoresis buffer (90 mmol/l tris-boric acid pH 8.3 and 2.5 mmol/l EDTA) at 50 V overnight.
3. At the end of electrophoresis, the gel membrane was stained with ethidium bromide (30~40 min), at which time the DNA could show fluorescent bands when irradiated with UV light.
4. Then we can use an absorbance meter for quantitative determination or compare the length of the fluorescent bands with a standard tube to obtain semi-quantitative results.
5. One enzyme unit means that 50% of 0.26ug superhelical DNA can be converted to unspun DNA within 30 min, and two enzyme units are used for each assay.
6. Isolation and preparation of type II DNA topoisomerases
(1) Human type II enzyme can be isolated from peripheral leukemia cells obtained from leukemia patients, e.g., chronic lymphocytic leukemia patients.
(2) The isolated enzyme is then purified by polyethylene glycol, followed by hydroxyapatite column chromatography, heparin-agarose column chromatography, and hexaminoagarose affinity chromatography.
(3) Staining with Coomassie blue was performed to visualize three bands of 142,000, 132,000 and 114,000 dalton.
7 Preparation of P4 superhelical DNA
