PULLDOWN PROTOCOL
Sergio Carilla
OBTAINING THE FUSSION PROTEIN
1. Grow bacteria expressing the protein until O.D = 0.4-0.6 in 100 ml (LB
medium) at 37 ºC. This volume can vary depending on the fusion protein of
interest.
2. Add IPTG (Sigma) to a final concentration of 100M and the incubation
continues for 2 h. (For optimal results try first growing in 3 ml LB and adding
IPTG for 0,1,2 or 3 hours. Then run a gel and test the protein levels by Comassie
staining).
3. Centrifuge 5 min 4000rpm RT
4. Resuspend the pellet in 5 ml of cold PBS 1X with protease inhibitor 1X.
5. Sonication 10 sec on 10 sec off 5 cycles until the solution clears up.
6. Centrifugation 8000g 10 min 4ºC.
7. Add 50 l of Glutathione Sepharose Bed (GE Healthcare) to supernatant of the
previous step. This beads have to be washed previously to eliminate ethanol
traces.
8. Mix at RT for 20 min.
9. Centrifugation 1 min 1000g RT for pull down the beds.
10. The beds are resuspended in 500 l of cold PBS 1X and transferred to protein
lobind eppendorf 1,5 ml tube.
11. Incubate 10 min 4ºC
12. Centrifugation 1 min 1000g 4ºC.
13. Repeat steps 10, 11 and 12 (3 washing steps).
14. Resuspend the beds in 150 l PBS 1X.
15. To test the fussion protein load 10 or 20 l of step 14 in a 10% polyacrilamyde
gel.
THE PULLDOWN
1. 2 x106-5x106 Dictyostelium or human cells are harvested by centrifugation at
1000g 10min RT. These cells contain the putative protein interactant (for
example fused to GFP).
2. Resuspend the pellet in 500 l of STET+T buffer (10mM Tris PH 8, 1mM
EDTA PH8, 150 mM NaCl, 5mM DTT, 1% Triton, protease inhibitor cocktail
1X) and incubate 30 min 4ºC.
3. Centrifugate 1 min 14000rpm 4ºC.
4. Incubate the supernatant with 30 l of beds obtained in step 14 for 1h 4ºC
5. Centrifuge 2 min 1000g 4ºC
6. Wash the beads 3 times with 500 l of STET+T buffer incubating 10 min 4ºC
between centrifugation steps (1000 g 4ºC)
7. Resuspend in 30 l protein loading buffer and load 2 gels, 15 l per gel: One for
comassie staining and second for western blotting analyses to detect the
interacting protein. Another way to do this is to load in the same gel the whole
sample, tranfer the gel to a membrane and satin with Ponceau red prior to block
the membrane.
ATTENTION:
 WORK, WHEN POSSIBLE, AT 4ºC FOR KEEPING THE PROTEIN
NATIVE
 TIPS MUST BE CUTTED TO ALLOW THE BEDS TO GET INTO
THEM
 BACTERIAL LYSIS CAN BE IMPROVED WITH LISOZYME (100
g/5ml).