DNMT ACTIVITY CUANTIFICATION KIT
PROTOCOL for ADIPOSE TISSUE PROTEINS
• EDM1, EDM2, EDM3 were prepared following manual instructions.
• The amount of protein obtained in the nuclear extraction of this kind of tissue was very low. Therefore, we decided to use 20 g of nuclear extract and complete with a total volume of 100 L/well. (After different probes we saw that we need this amount of protein, and we safe the proportion, 1-5 L of nuclear extract and 40-45 L of EDM3, completing with 100 L/well.)
• For blank and positive control wells, the volume was changed to 100 L/well.
• The strip-well microplate was covered with the adhesive film and was incubated in an oven at 37ºC with continued shaking during 3 hours.
• The washes were done following the instructions in all steps.
• EDM5 were prepared in a dilution rate of 1:500 and incubated in the same conditions that in the previous step (oven at 37ºC with continued shaking during 1 hour).
• EDM6 were prepared in a dilution rate of 1:1000 and incubated in the same conditions that in the previous step (oven at 37ºC with continued shaking during 30minutes).
• EDM7 were prepared in a dilution rate of 1:2000 and incubated in the same conditions that in the previous step (oven at 37ºC with continued shaking during 30minutes).
• 100 L/well of EDM8 were added and the microplate was incubating at room temperature away from direct light about 30 minutes.
• EDM9 were added to stop the enzyme reaction.
• Activity calculation was done following the instructions.
PROTOCOL for ADIPOSE TISSUE PROTEINS
• EDM1, EDM2, EDM3 were prepared following manual instructions.
• The amount of protein obtained in the nuclear extraction of this kind of tissue was very low. Therefore, we decided to use 20 g of nuclear extract and complete with a total volume of 100 L/well. (After different probes we saw that we need this amount of protein, and we safe the proportion, 1-5 L of nuclear extract and 40-45 L of EDM3, completing with 100 L/well.)
• For blank and positive control wells, the volume was changed to 100 L/well.
• The strip-well microplate was covered with the adhesive film and was incubated in an oven at 37ºC with continued shaking during 3 hours.
• The washes were done following the instructions in all steps.
• EDM5 were prepared in a dilution rate of 1:500 and incubated in the same conditions that in the previous step (oven at 37ºC with continued shaking during 1 hour).
• EDM6 were prepared in a dilution rate of 1:1000 and incubated in the same conditions that in the previous step (oven at 37ºC with continued shaking during 30minutes).
• EDM7 were prepared in a dilution rate of 1:2000 and incubated in the same conditions that in the previous step (oven at 37ºC with continued shaking during 30minutes).
• 100 L/well of EDM8 were added and the microplate was incubating at room temperature away from direct light about 30 minutes.
• EDM9 were added to stop the enzyme reaction.
• Activity calculation was done following the instructions.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Miss. Ana Jadraque
Verified customer
提交于 Jun 25 2014