Flag-HA double tag IP
实验概要
Isolate the unknown protein that could interact with bait from cell lysate, for potential Mass spectrometry.
主要试剂
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
实验步骤
Havest the cells by centrifugation at 1,000 rpm for 3 min. Wash the cells with ice cold PBS twice.
Resuspend the cell pellet with 5 volumes of Flag lysis buffer. Incubate on ice for 20 min.
Centrifuge at max speed at 4 °C for 30 min. Save the supernatant.
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.
If you do SILAC experiment, mix the two samples at equal protein amount at this time.
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.
Incubate Flag-beads with the lysate on a rotator at 4 °C overnight (10 rpm).
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.
Wash the beads with 1 ml Flag lysis buffer. Repeat the wash for 5 times.
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.
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.
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.
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.
