Preparation for Cell Lysis
Obtain the mammalian expression vector for homo sapien ESR2 from Addgene. Assemble the vector to code for the proteins snail, slug and twist. Transfect HEK293 cells with the modified plasmids in petri dishes. Allow the cells to grow for 1-2 days so they will express the proteins.
Transfer the culture medium to a centrifuge tube to separate it from the cells. Add 1mL of phosphate buffered saline (PBS) to the cells and swirl to mix. Separate the PBS from the cells and combine the PBS with the culture medium. Trypsinize the cells with 300 µL of trypsin to and incubate them at 37℃ to dissociate the cells from the petri dishes. (If the cells appear to be clustered together, break them apart by gently pipetting them until they are separated.) Add the cells to the centrifuge tube and centrifuge. Discard the supernatant, add 2 mL of PBS and vortex it to suspend the mixture. Obtain 20 µL of the mixture and place it on ice for future use to count the cells. Cell Lysis Centrifuge the cells at 4℃ and discard the supernatant. Add 600 µL of lysis buffer to the centrifuge tube and resuspend it using the vortex. Allow the cell suspension to lyse by placing it on ice for 30 minutes. Centrifuge the mixture and transfer the supernatant to a separate centrifuge tube to isolate the lysates. Note: Keep the …show more content…
Centrifuge for 5-10 seconds and discard the supernatant. Add lysis buffer with a volume equal to that of the pellet and invert the tube to resuspend the Sepharose beads. Repeat this part of the procedure two additional times to obtain a mixture with 50% PAS in lysis buffer. Dilute the lysate (with a volume equal to 8 * 10⁵ cells) to 300 µL in a 1.5 mL microfuge tube and add 50 µL of the washed 50% PAS. Rock the mixture at 4℃ for 1 hour to keep it suspended. Centrifuge the mixture for 60 seconds and transfer the supernatant to a separate centrifuge tube to isolate the cleared
Obtain the mammalian expression vector for homo sapien ESR2 from Addgene. Assemble the vector to code for the proteins snail, slug and twist. Transfect HEK293 cells with the modified plasmids in petri dishes. Allow the cells to grow for 1-2 days so they will express the proteins.
Transfer the culture medium to a centrifuge tube to separate it from the cells. Add 1mL of phosphate buffered saline (PBS) to the cells and swirl to mix. Separate the PBS from the cells and combine the PBS with the culture medium. Trypsinize the cells with 300 µL of trypsin to and incubate them at 37℃ to dissociate the cells from the petri dishes. (If the cells appear to be clustered together, break them apart by gently pipetting them until they are separated.) Add the cells to the centrifuge tube and centrifuge. Discard the supernatant, add 2 mL of PBS and vortex it to suspend the mixture. Obtain 20 µL of the mixture and place it on ice for future use to count the cells. Cell Lysis Centrifuge the cells at 4℃ and discard the supernatant. Add 600 µL of lysis buffer to the centrifuge tube and resuspend it using the vortex. Allow the cell suspension to lyse by placing it on ice for 30 minutes. Centrifuge the mixture and transfer the supernatant to a separate centrifuge tube to isolate the lysates. Note: Keep the …show more content…
Centrifuge for 5-10 seconds and discard the supernatant. Add lysis buffer with a volume equal to that of the pellet and invert the tube to resuspend the Sepharose beads. Repeat this part of the procedure two additional times to obtain a mixture with 50% PAS in lysis buffer. Dilute the lysate (with a volume equal to 8 * 10⁵ cells) to 300 µL in a 1.5 mL microfuge tube and add 50 µL of the washed 50% PAS. Rock the mixture at 4℃ for 1 hour to keep it suspended. Centrifuge the mixture for 60 seconds and transfer the supernatant to a separate centrifuge tube to isolate the cleared