FLAG affinity purification and mass spectrometry

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

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