Affinity purification of biotinylated proteins

1. Freeze treated plant material in liquid nitrogen and grind into a fine powder (make sure it does not thaw)

2. Transfer an adequate amount of powder into a 15 ml tube (up to 3 ml if PD-10 desalting columns are used for biotin depletion)

3. Add 2/3 volume ice cold extraction buffer (2 ml for 3 ml plant material)

4. Incubate on a rotor wheel at 4°C until the plant material is resuspended (10 min or more)

5. Add 1µl Lysonase (Millipore) and incubate for 15min on the rotor wheel at 4°C

6. Split sample into 2-3 1.5 ml reaction tubes and sonicate 4 x 30sec on high settings in an ice bath with 1.5 min breaks (Bioruptor UCD-200, Diagenode)

7. Centrifuge samples for 15min at 4°C, 15000g and transfer the supernatants into a fresh tube

8. Deplete free biotin using PD-10 desalting columns (GE healthcare):

9. Pour storage buffer off the column and cut the sealed bottom

10. Fill the column 5x with equilibration buffer (extraction w/o complete and PMSF) and allow the buffer to enter the bed completely (25ml)

11. Load 2.5ml of the supernatant onto the column and let it enter the bed, (if less than 2.5ml, add extraction buffer to a final volume of 2.5ml and let it enter the bed completely)

12. Place a new 5ml tube under the column and elute with 3.5ml equilibration buffer

13. Measure the protein concentration by Bradford (BioRad protein assay). Attention! Samples must be diluted at least 1:5 as some buffer components can interfere with the assay

14. Transfer the desired amount of protein extract into a new 5ml LoBind tube containing 200µl streptavidin beads (e.g. MyOne Streptavidin C1 (Invitrogen)) that were pre-washed with extraction buffer. Ideally the same amount of total protein is used for all samples in one experiment.

15. Add complete and PMSF to each sample to reach a final concentration of 1x and 1 mM, respectively

16. Incubate on a rotor wheel at 4°C (a few hours or over night)

17. Remove the supernatant and wash the beads 2 x with 1 ml ice cold extraction buffer by incubating on a rotor wheel at 4°C for 8 min and separating the beads from the solution. Transfer the beads to a fresh tube during the first wash.

18. Wash the beads 1 x with cold 1 M KCl

19. Wash the beads 1 x with cold 100 mM Na2CO3

20. Wash the beads 1 x with 2M Urea in 10 mM Tris pH 8 at room temperature

21. Wash the beads 2 x with cold equilibration buffer

22. Use the samples directly for further MS sample preparation (tryptic digest) or remove the remaining liquid and freeze at -80°C until further use

Buffers and solutions:
Solutions for the final wash steps should be made with ultra pure (HPLC purified) water for MS analysis

• Extractions buffer: 50mM Tris pH 7.5, 150mM NaCl, 0.1% SDS, 1% Triton-X-100, 0.5% Na-deoxycholate, 1mM EGTA, 1mM DTT, 1x complete, 1mM PMSF

• Equilibration buffer: 50mM Tris pH 7.5, 150mM NaCl, 0.1% SDS, 1% Triton-X-100, 0.5% Na-deoxycholate, 1mM EGTA, 1mM DTT

• 1 M KCl

• 100 mM Na2CO3

• 2M Urea in 10 mM Tris pH 8 (prepare fresh)