Chromosome immunoprecipitation method and kit for DNA binding proteins in eukaryotic cells
A technology of co-immunoprecipitation and eukaryotic cells, which is applied in the field of chromosomal co-immunoprecipitation of DNA-binding proteins in eukaryotic cells and related kits, which can solve the problems affecting analysis and detection, different binding abilities, different target DNA, etc. question
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Embodiment 1
[0111] 1. Get 10 grams of neural brain tissue from 8 mice, and wash with PBS (137mM NaCl; 2.7mM KCl; 10mM NaCl 2 HPO 4 12H 2 O; 2 mM KH 2 PO 4 ; 0.1% by weight PMSF) was dispersed into 990ml cell suspension by pipetting and washing.
[0112] 2. Add 10ml formaldehyde to a final concentration of 1% (v / v), add 1ml 2M glycine to neutralize the cross-linking effect after 5 minutes, centrifuge and discard the supernatant, wash with PBS, centrifuge and discard the supernatant, the number of eukaryotic cells is about 10 8 indivual.
[0113] 3. Add 500 μl 1% SDS lysate and place on ice for 10 minutes.
[0114] 4. Ultrasound to disrupt cells, the ultrasonic program is as follows: 30S on, 30S off, 9 ultrasonic cycles, ultrasonic frequency 60kHz. After the sonication is over, centrifuge to take the supernatant and discard the precipitate.
[0115] 5. Pretreat the agarose beads coated with protein A, protein G and protein L (use the first washing solution to wash the agarose beads ...
Embodiment 2
[0122] 1. Take 10 grams of liver tissue from 2 mice, wash with PBS (137mM NaCl; 2.7mM KCl; 10mM NaCl 2 HPO 4 12H 2 O; 2 mM KH 2 PO 4 ; 0.1% by weight PMSF was dispersed into 999ml cell suspension by pipetting and washing.
[0123] 2. Add 1ml of formaldehyde to a final concentration of 0.1% (v / v), add 1ml of 2M glycine to neutralize the cross-linking effect after 10 minutes, centrifuge and discard the supernatant, wash with PBS, centrifuge and discard the supernatant, the number of eukaryotic cells is about 5×10 7 indivual.
[0124] 3. Add 500 μl 1% SDS lysate and place on ice for 15 minutes.
[0125] 4. Ultrasonic disrupts the cells. The ultrasonic program is as follows: 20S on, 45S off, 8 ultrasonic cycles, and the ultrasonic frequency is 40kHz. After the sonication is over, centrifuge to take the supernatant and discard the precipitate.
[0126] 5. Pretreat the agarose beads coated with protein A, protein G and protein L (wash the agarose beads for 1 hour with the fi...
Embodiment 3
[0133] 1. Take the spleen tissues of 30 mice (10 grams in total), wash with PBS (137mM NaCl; 2.7mM KCl; 10mM NaCl 2 HPO 4 12H 2 O; 2 mM KH 2 PO 4 ; 0.1% by weight PMSF) was dispersed into 950ml cell suspension by pipetting and washing.
[0134] 2. Add 50ml formaldehyde to a final concentration of 5% (v / v), add 1ml 2M glycine to neutralize the cross-linking effect after 20 minutes, centrifuge and discard the supernatant, wash with PBS, centrifuge and discard the supernatant, the number of eukaryotic cells is about 15×10 7 indivual.
[0135] 3. Add 500 μl 1% SDS lysate and place on ice for 8 minutes.
[0136] 4. Ultrasound to disrupt cells, the ultrasonic program is as follows: 40S on, 60S off, 15 ultrasonic cycles, ultrasonic frequency 20kHz. After the sonication is over, centrifuge to take the supernatant and discard the precipitate.
[0137] 5. Pretreat the agarose beads coated with protein A, protein G and protein L (use the first washing solution to wash the agarose...
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