FLAG-tagged Purifications from Yeast for Mass Spectrometry

1. Grow 20mL 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 1L of YPD.  Grow at 30ºC until the cells reach OD₆₀₀ ~1.0 (5-6 hours). (For low abundance proteins, increase culture volume. Can increase up to 6L or more.)

3. Harvest cells by centrifugation at 3500rpm in large bottle for 10 minutes at 4ºC. Pour off supernatant.

4. Wash cells in 40 mL ice-cold water.  Transfer to a 50mL conical tube. Centrifuge at 3500rpm for 4 minutes at 4ºC. Pour off supernatant.

5. Resuspend cells in volume of lysis buffer + 1mM PMSF+1x yeast protease inhibitors (Sigma) + 1x phosphatase inhibitors (PhosSTOP, Roche/Sigma) equivalent to the approximate size of the pellet (e.g. If pellet is ~5mL, resuspend in 5mL of buffer). 

6. Freeze dropwise in liquid nitrogen.  Transfer to 50mL conical tube for storage at -80ºC until proceeding with the purification.

7. In the cold room, fill a coffee grinder half way with dry ice.  Add cell pellets and grind for 1 minute.

8. Pour/scrape the ground cells in to a 14mL centrifuge tube.  Allow the dry ice to evaporate. Once the dry ice has evaporated and the extract is liquid, add NP-40 to a final concentration of 0.4% and invert 10 times to mix well.    

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

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

During this step, wash anti-FLAG magnetic beads as follows:

-Pipet 100 µL (per IP) of 50/50 bead slurry in to an eppendorf tube (for 2 IPs, use 200 µL). If doing more than 2 IPs, perform bead washes in 15mL conical tube with 3mLs of wash buffer each time.  

      -Add 1 mL of lysis buffer (w/o PMSF, protease and phosphatase       inhibitors) to the tube and mix by inverting 5-6 times (do not vortex!).

      -Put tube in the magnetic stand and allow beads to collect against the wall       of the tube. 

      -Remove supernatant.

      -Repeat wash with 1mL of lysis buffer, mix by inversion, magnet, remove       supernatant.

-After final wash, resuspend beads in an equivalent amount of lysis buffer  to generate a 50/50 slurry.

11. Remove supernatant to a 15mL conical tube.  Remove 100 µL of “total” for analysis by western.

12. Perform a Bradford assay to determine total protein concentration for each sample. Calculate the total protein in the extract with the lowest concentration. Normalize the other lysates such that they have equivalent amounts of total protein in the same volume.  Use lysis buffer to increase volume if necessary. (This normalization step improves ability to compare levels of background proteins that may non-specifically precipitate in each IP.)  

13.  Add 100 µL of 50/50 slurry of beads to each extract.  Rotate at 4ºC for 3 hours.

14. Place tubes in the magnetic stand.  Remove supernatant (flow-through) and save for 100 µL for analysis. 

15. Wash beads 3x with lysis buffer: Add 5 mL of lysis buffer + 0.5% NP-40, rotate for 5 minutes at room temperature, place in magnet and remove supernatant.

16. After the last wash, transfer beads to an eppendorf tube using 500 µL of   lysis buffer + 0.5% NP-40. Wash tube and pipet tip with an additional 500 µL of buffer and pool with the remaining sample. Separate beads with magnet and remove supernatant.

Elution option #1: FLAG peptide

17. Prepare 0.2mg/mL 3xFLAG peptide in lysis buffer from 5mg/mL stock of 3xFLAG peptide.  Prepare fresh, just before elution. 

18. Add 100 µL of elution buffer with FLAG peptide to tube. Rotate at 4ºC for 30 minutes.

19. Place tubes in magnetic stand and remove eluate to a new tube. 

20. Repeat elution by adding 100 µL of elution buffer with FLAG peptide to tube. Rotate at 4ºC for 30 minutes.

21. Place tubes in magnetic stand and remove eluate to another tube (a different tube than eluate #1). 

22. Add 20 µL of 5X SDS loading buffer to each eluate and boil for 3 minutes.  Load 30 µL on SDS-PAGE for silver staining and 10 µL for western blotting.    

Elution option #2: Glycine, low pH

17. Elute proteins with 0.1M glycine pH 3.0: Add 100 µL of 0.1M glycine to beads and incubate at room temperature for 15 minutes.  During the incubation, mix beads every 2-3 minutes by flicking the tube.

18. Separate beads and remove eluate to a new tube containing 10 µL of neutralization buffer. 

19. Add 20 µL of 5X SDS loading buffer and boil for 3 minutes.  Load 30 µL on SDS-PAGE for silver staining and 10 µL for western blotting.    

Lysis Buffer

100mM HEPES pH 8.0

20mM MgOAc

10% glycerol

10mM EGTA

0.1mM EDTA

FLAG peptide elution buffer

0.2mg/mL FLAG peptide in lysis buffer (diluted from 5mg/mL stock of FLAG peptide)

Glycine Elution Buffer

0.1M glycine pH 3.0

Neutralization Buffer

0.5M Tris-HCl pH 7.4

1.5M NaCl