FlagHAdoubletagIP

實驗概要

Isolate the unknown protein that could interact with bait from cell lysate, for potential Mass spectrometry.

主要試劑

50mM Tris, pH8.0

20 mM glycerol b-phosphate, pH 7.5

20 mM NaF

0.5 mM sodium Orthovannadate

5 mM sodium pyrophosphate

137mM NaCl

1mM EDTA

1% Triton X100

10% glycerol

實驗步驟

1. Havest the cells by centrifugation at 1,000 rpm for 3 min. Wash the cells with ice cold PBS twice.

2. Resuspend the cell pellet with 5 volumes of Flag lysis buffer. Incubate on ice for 20 min.

3. Centrifuge at max speed at 4 °C for 30 min. Save the supernatant.

4. Determine  the protein concentration by Bradford reagent. Dilute the lysate to 2  mg/mL (or even 1 mg/mL if possible) with Flag lysis buffer.

5. If you do SILAC experiment, mix the two samples at equal protein amount at this time.

6. Wash  Flag-beads (Sigma) with Flag lysis buffer twice. Add the beads into the  lysate. For 10 mL lysate, add 30 mL beads volumn of Flag-beads. For 50  mL lysate, add 70 mL.

7. Incubate Flag-beads with the lysate on a rotator at 4 °C overnight (10 rpm).

8. Next  morning, spin down the beads at 3,000 rpm for 10 min at 4°C. Remove  supernatant, and transfer the beads to 1.5 mL eppendorf tube.

9. Wash the beads with 1 ml Flag lysis buffer. Repeat the wash for 5 times.

10. Elute  the Flag-beads with 5 volumes of 0.5 mg/mL 3′Flag peptides (dilute in  Flag lysis buffer). Incubate at 4°C on the rotator for at least 6 hrs.

11. Spin  down the Flag-beads. Transfer the supernatant to a 0.5 mL eppendorf  tube. Add 20 mL beads volume of HA-beads (Sigma) to the lysate, and  incubate on the rotator at 4 °C overnight.

12. Next  morning, wash the HA-beads with Flag lysis buffer for 5 times. Transfer  the HA-beads to a PCR tube. Elute the HA-beads with 100 mL of 1 mg/mL  HA-peptide (diluted in Flag lysis buffer). Incubate on the rotator at 4  °C for at least 6 hrs.

13. Spin  down, save the supernatant. Add 15 mL of 4′SDS loading buffer, and boil  to about 60 mL. Load the sample to a gradient gel and proceed to silver  staining.