The main biochemical feature of apoptosis is the condensation of chromatin, the cleavage of chromatin DNA at the junction between nucleosome units, the formation of large DNA fragments of 50-300 kbp long, or oligonucleosides of integer multiples of 180-200 bp. The acid fragment appears as a ladder ladder on gel electrophoresis. After the cells were treated, the DNA was isolated and purified by a conventional method, and subjected to agarose gel and ethidium bromide staining, and a typical DNA ladder was observed in the apoptotic cell population. If the amount of cells is small, DNA can be labeled with 32P-ATP and deoxyribonucleotide terminal transferase (TdT) after separation and purification of DNA, followed by electrophoresis and autoradiography to observe DNA ladder in apoptotic cells. form. 1. Determination of DNA fragments of macromolecules In the early stage of apoptosis, the chromosome breaks into a large DNA fragment of 50-300 kbp. All double-stranded DNA molecules over a certain molecular weight have the same rate of migration in an agarose gel. When the double helix radius of the linear DNA exceeds the gel radius, the resolution limit is reached. At this point, the gel no longer sifts the DNA by the molecular weight. The DNA passes through the gel like a bend, with one end pointing toward the pole of the electric field. This mode of migration is called "crawling." Therefore, large fragments of 50-300 kbp long DNA generated early in apoptosis cannot be separated by ordinary agarose gel electrophoresis. Pulse electrophoresis is often used to solve this problem satisfactorily. This method is to apply an orthogonal alternating pulse electric field to the gel. Whenever the direction of the electric field changes, large DNA molecules stagnate in the crawler tube until the new electric field is axially reoriented before moving forward. The greater the molecular weight of the DNA, the longer it takes for this rearrangement. When the DNA molecules change direction for less than the electrical pulse period, the DNA can be separated by its molecular weight. 2. DNA Ladder determination method: 1 harvested cells (1X107) precipitated; 2 cell lysate: 13000 rpm '5 min, the supernatant was collected; 31% SDS and RnaseA (5 mg/ml) 56 ° C, 2 h; 4 proteinase K (2.5mg/ml) 37 ° C, 2h; Shanghai Chuangsai Technology provides 39450-01-6, proteinase K, Proteinase K, for molecular biology, ≥ 30 units / mg protein, commodity number: C16- P10706-25mg, the price is 165 yuan. Precipitate DNA with 51/10 volume of 3M sodium acetate and 2.5 volumes of cold absolute ethanol, overnight at 4 °C, 14000 rpm '15 min; Shanghai Chuangsai Technology provides 4418-26-2, sodium dehydroacetate, 99%, commodity number: D16-1019891-100g, the price is 155 yuan. 6 Finally, the precipitate is dissolved in TE buffer, and DNA Loading Buffer is added; 71.2% agarose gel electrophoresis, EB staining and photographing. 3. Flow cytometry analysis of DNA content of apoptotic cells method: 1 Collect cells, 70% cold ethanol (in PBS) fixed at 4 ° C overnight, washed with PBS; Shanghai Chuangsai Technology provides recombinant human RNase A, Ribonuclease A, RNASE1 protein (His tag), Ribonuclease, RNase A family, 1 (pancreatic ) Protein, product number: C86-134-68-50ug, price 2210 yuan. 21000rpm'10min RNase A (0.5mg/ml) was digested at 37°C for 30min PI (50mg/ml) staining, protected from light for 15min at room temperature; Shanghai Chuangsai Technology provided 957054-30-7, GDC-0941GDC0941>98%, effective PI3Kα /δ inhibitor, commodity number: C84-4843-10mg, price 931 yuan. 3FACScan analyzes DNA diploid formation and cell cycle changes. 4. ApoAlertTM LM-pcr Ladder Assay (CLONTECH) Advantages: high sensitivity, suitable for detecting a small number of samples, a small number of apoptotic cells. Such as clinical biopsy. CANSPEC quality provides excellent performance, its excellent quality to meet the various needs of your different experiments, spot, product details, scientific instruments, laboratory supplies, Shanghai Chuangsai, mass products special promotions, welcome to inquire! Table Series,College Classroom Desk,Wooden Reading Table,Desks With Wheel AU-PINY FURNITURE CO., LTD , https://www.aupinyjm.com
DNA fragmentation detection