FLAG affinity purification for mass spectrometry

Lab. Luciano Di Croce

1. Grow PC3 cells stably expressing FLAG tagged constructs in 150 mm dishes until they reach 75% confluency. For each condition 3-4 150 mm dishes will be necessary to obtain 50-60 million cells.

2. Remove growth medium and rinse the cells twice with PBS. Discard the PBS after each rinse.

3. Add 3 ml of cold PBS containing protease / phosphatase inhibitors to each plate. Gently scrape the cells from the plate and collect them in a 15 ml tube. Combine cells from 3-4 dishes of the same condition in one tube. Spin the tubes at 300 x g for 5 minutes at 4ºC to obtain cell pellet.

4. Resuspend the cells in 1.5 ml cold Lysis Buffer containing protease / phosphatase inhibitors and incubate for 30 minutes on a rotating wheel at 4ºC.

5. Sonicate the lysate for 3 cycles (10sec ON / 30sec OFF) in a Bioruptor Diagenode.

6. Clarify the lysate by centrifugation at 15000 x g for 30 minutes at 4ºC.

7. Quantify the amount of protein in the lysate using the Bradford assay.

8. Aliquot 5 mg of cell lysate to proceed with FLAG immunoprecipitation, and keep an aliquot of 10% of the volume as input control for Western blot analysis.

9. Thoroughly resuspend the ANTI-FLAG M2 affinity gel (SIGMA) and transfer 100 µl for each immunoprecipitation to a centrifuge tube. Centrifuge at 5000-8000 x g for 30 seconds at 4ºC. Wait for 1-2 minutes for the resin to settle and remove the supernatant very carefully

10. Wash the ANTI-FLAG M2 affinity gel twice with 1 ml TBS. Centrifuge at 1000 x g for 5 minutes at 4ºC and discard the supernatant.

11. Bring the cell lysate obtained in step 8 (5 mg) to 1 ml with Lysis Buffer plus protease / phosphatase inhibitors, and add it to the washed ANTI-FLAG M2 affinity gel. Incubate for 3 hours on a rotating wheel at 4ºC and centrifuge at 1000 × g for 5 minutes at 4ºC.

12. Wash the resin beads three times with 1 ml Lysis Buffer, followed by two washes with 1 ml of TBS. Collect the resin after each wash by centrifugation at 1000 x g for 5 minutes at 4ºC.

13. Add 100 µl of Elution Buffer and elute the complex by shaking at 1000 rpm for 30 minutes at room temperature, followed by centrifugation at 5000-8000 x g for 30 seconds at room temperature. Transfer the supernatant to a fresh tube. Perform two rounds of elution for maximum recovery.

14. Aliquot 10% of the eluate for analysis. Run the aliquots of eluate and input samples in a SDS polyacrylamide-gel and check the efficiency of the immunoprecipitation by Western blotting.

15. Send the remaining eluated sample for mass spectrometry analysis. Samples in the present study were trypsin-digested and analyzed mass spectrometry by the Proteomic Unit at the CRG/UPF.

Lysis Buffer:

50 mM Tris-HCl pH 7.5; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 0.5 % Triton x100. Add protease / phosphatase inhibitors immediately before use.

TBS:

50 mM Tris-HCl pH 7.5; 150 mM NaCl

Elution buffer:

6M Urea; 200 mM NaHCO₃

OPTION: Alternative elution buffers could be used, as long as they are compatible with the ANTI-FLAG M2 resin and downstream processing of the eluted complex for mass spectrometry.

Protease / phosphatase inhibitors:

1x Complete EDTA-free protease inhibitor cocktail (Roche); 1 mM PMSF; 1 mM Na₃VO₄; 10 mM β-Glycerophosphate