核心提示：1) Aliquot 5 X 106 total cells in 5-8 ml 2% media.2) Add 30 µl
3H-thymidine.3) Incubate for approximately 16 hour1) Aliquot 5 X 106
total cells in 5-8 ml 2% media.2) Add 30 µl 3H-thymidine.3) Incubate for
approximately 16 hours under normal growth conditions.4) Spin down cells
and wash 2-3 times with PBS to remove any unincorporated label.5)
Resuspend cells at 5 X 105/ml in serum free media.6) Aliquot
approximately 400 µl of cell suspension into each well of a 24 well
plate.7) Treat cells .8) Pellet cells gently and count
supernatant.–> supernatant = A9) Resuspend pellet in lysis
buffer.10) Microfuge cell suspension for 15 minutes at maximum speed.11)
Collect pellet and supernatant and count both.–> supernatant =
Bpellet = C12) To determine the percent fragmentation use the following
equation:–> /Lysis buffer
核心提示：OutlineImmunoprecipitation is a technique that permits the
purification of specific proteins for which a antibody has
核心提示：In order to decrease the amount of nonspecific staining, it is
often necessary to preabsorb primary and secondary ant
Immunoprecipitation is a technique that permits the purification of
specific proteins for which a antibody has been raised. This primary
antibody is either already bound to agarose or can be bound to the
protein A/agarose beads during the procedure in order to physically
separate the antibody-antigen complex from the remaining sample.
In order to decrease the amount of nonspecific staining, it is often
necessary to preabsorb primary and secondary antibodies to yeast cells
lacking the antigen prior to use. A 1:1 mixture of fixed yeast whole
cells and spheroplasts are used for this purpose.
Supplies / Equipment
- sterile 1.5 µl microfuge tubes
- sterile pipette tips
- waterbath 100°C
- microfuge 4°C
- rocking platform 4°C
- rocking platform 25°C / room temperature
- Grow cells in 200 mls of YPD at 30oC to an OD 600 of 1.
- Add formaldehyde to the 200 ml culture to a final concentration of
3.7%. Fix for 1 hour at room temperature with shaking.
- Centrifuge cells at 3000 X g. Resuspend in 200 mls Solution A.
- Pellet cells as above and resuspend in 4 mls Solution A. Reserve 2
mls of the cell suspension.
- Spheroplast the remaining 2 ml of the cell suspension by addition of
beta-mercaptoethanol to 0.1%, glusulase to 0.05%, and zymolyase to
60 ug/ml. Incubate 1 hour at 37oC.
- Pellet spheroplasts as above. Resuspend in 10 mls Solution A and
- Combine the suspension of whole cells with the spheroplasts and
pellet as above. Resuspend in 10 mls of PBS.
- Put 200 ul of the antibody to an eppendorf tube. Add 0.8 mls of PBS.
- Pellet 1 ml of the cell suspension and discard the PBS supernatant.
Add the antibody solution to the cell pellet, gently resuspend the
cells, and incubate for 1 hour on ice.
- Pellet cells, giving an antibody supernatant. Add this to a cell
pellet prepared as in step 9. Be very careful not to get
supernatants mixed up!
- Repeat steps 9 + 10 seven times, or as often as necessary to
decrease the background.
- Pellet cells. Save antibody supernatant, which is now diluted 1:5
from its initial concentration. These antibodies can be stored at
4oC for several weeks or at -70oC for longer periods.