Co-immunoprecipitation of Chromatin Proteins from Yeast Extracts

Set up antibody binding to magnetic beads:

A.    Mix beads well and remove 20 µL bead slurry (per IP) to a 1.5mL tube.

B.    Place bead tube in magnetic stand. Allow beads to collect on side of tube. Remove supernatant.

C.    Wash 3X in 1 mL TBS.

D.    Add 200 µL TBS to beads and appropriate amount of antibody (1-5µg). Incubate O/N, rotating @ 4°C.

E.    Place beads in magnetic stand and remove supernatant.

F.    Wash 1X in 1 mL TBS.

G.   Resuspend beads in 40 µL TBS per IP.

Add 35 µL to each IP

·       Increase volume dependent on total number of IP’s – This is for 1 IP

1. Grow 5mL of appropriate yeast strains overnight in YPD at 30ºC.

2. The next morning, measure the OD₆₀₀ of the culture.  Dilute to OD₆₀₀ = 0.2 in 25mL of YPD.  Grow at 30ºC until the cells reach OD₆₀₀ ~1.0 (5-6 hours).

*This volume of cells is sufficient for one IP from one culture—scale up for more than one IP.

3. Harvest cells by centrifugation at 3500rpm for 4 minutes at 4ºC. Pour off supernatant.

4. Wash cells in 25mL ice-cold water.  Centrifuge at 3500rpm for 4 minutes at 4ºC. Pour off supernatant.

5. Resuspend cells in 1.2mL of cold lysis buffer* + 1mMPMSF + 1x protease inhibitor cocktail (Sigma; stock is 1000x).  *Add PMSF and protease inhibitors just before use. 

6. Transfer cells to 2.0mL screw cap tube containing acid-washed glass beads for lysis.  Place tubes in the BeadBug in the cold room.  Shake on maximum speed for 20 sec, place on ice for 30 sec.  Repeat 3 times (4 times total).

7. Transfer lysate to a clean eppendorf tube by pipetting off the top.

8. Sonicate whole cell extracts 3x for 30 sec each at 15% output with 1 minute on ice between each sonication. 

9. Clarify extracts by centrifugation at 5000rpm for 10min at 4ºC.

10. Remove supernatant to a new eppendorf tube.  This is the clarified extract you will use for the IP.  Set aside 50 µL as the “total” for western blots.

11. Transfer 1mL of extract to new eppendorf tube**.  Add antibody plus beads to each tube (as described above).  Rotate at 4ºC for 3 hours (or overnight).   

      **Alternatively, if comparing IPs between different mutant strains or conditions, perform a Bradford assay to determine total protein concentration for each sample. Calculate the total protein in the lysate with the lowest concentration. Normalize the other lysates such that they have equivalent amounts of total protein in 1mL final volume.  Use lysis buffer to increase volume if necessary.

12. Place tubes in the magnetic stand.  Remove supernatant. 

13. Wash beads 3x with lysis buffer: Add 1 mL of buffer, rotate for 5 minutes at room temperature, place in magnet and remove supernatant. 

14. After last wash, add 50 µL of 1x SDS-PAGE loading buffer.  Rotate for 10 minutes at room temperature. 

15. Place tubes in the magnetic stand and remove supernatant to a new tube. 

16.  Add 10 µL of 5x SDS-PAGE loading buffer to the “total” sample and boil for 3 minutes. 

17. Load 10 µL of “total” samples and 25 µL of IP samples on to an SDS-PAGE gel for analysis by western blot. Load two identical gels to probe with both antibdodies for the coIP.   

Lysis Buffer                      Stock solution          Volume (for 200mL)

100mM HEPES pH 8.0             1 M                  20 mL

20mM magnesium acetate             1 M                  4 mL

10% glycerol                            100%                     20 mL

10mM EGTA (pH 8.0)              0.5 M                     4 mL   

0.1mM EDTA (pH8.0)               0.5 M                     40 µL

0.4% NP-40                             10%                      8 mL

 water                                                      144 mL

Alternate buffers:

IP Lysis buffer

50mM Tris-HCl pH 7.5

150mM NaCl

1mM EDTA

1% NP-40

IP Wash buffer

50mM Tris-HCl pH 7.5

300mM NaCl

1mM EDTA

1% NP-40