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Rong’s 293T suspension+mass spec

Protocols

Rong's 293T suspension cell+mass spec protocol

Grow 293T suspension cells in Freestyle medium (Invitrogen) plus 1% FBS and 1% Glutamine.

293T cells are thawed at 0.5-1.0 X 106 cells/ml. Cells are splitted to ～0.5 X 106 when they reach 2-3 X 106(normally every two days).

Transfection for 100ml cell culture:

  1. Mix 400ug PEI (1ug/ul, polyethyleneimine, Polysciences, Inc. Cat# 24765) and 5ml hybridoma (Invitrogen, Cat# 12045-084, prewarmed) and incubate 5min RT
  2. Mix 100ug DNA and 5ml hybridoma
  3. Mix PEI and DNA together and incubate for a further 10 min
  4. Add 3) directly to culture (about 1.5-2.0 X 106 cells/ml)
  5. Treat with 1mM HU for 16hrs before harvesting.
  6. Harvest cells at 2.5 days.

lyse cells (on ice)

  1. wash the cell pellet twice with PBS
  2. Resuspend with 2vol of cytoplasmic lysis buffer
  3. Add equal vol of cytoplasmic lysis buffer with 0.2% NP-40 (final 0.1% NP-40), 5min on ice
  4. Spin at 3,500g for 5min, keep supernatant “cytoplasmic fraction”
  5. Wash the pellet with 5vol of cytoplasmic lysis buffer
  6. Spin at 3,500g for 5min
  7. Add 2ml nuclear lysis buffer 2, resuspend and homogenize 20 up and down
  8. Measure volume and add Benzonase (Novagen, 25u/ul) 7ul/ml, rotating in cold room for 3hrs
  9. Spin at 13,200g for 5min, keep supernatant “chromatin fraction”

IP

  1. Dilute either cytoplasmic (4 folds) or chromatin (3 folds) fraction with washing buffer.
  2. Spin at 90,000g for 15min at 4 degree
  3. Filter the supernatant with PES syringe filter (Pall AP-4425)
  4. Add 30ul Flag beads (washed three times with 1ml washing buffer) per one sample
  5. Rotating in cold room for 4hrs
  6. Spin and wash with washing buffer 4 times at room temperature, rotating 10min for each wash
  7. Add 70ul 400ug/ml Flag peptide (Sigma) in TBS plus 5% glycerol, rotating in cold room for 1.5hrs
  8. Take out supernatant and check 10ul on 8-16%Tris-Glycine gel (Invitrogen)

Cytoplasmic lysis buffer: 10 mM Tris.HCl (pH 7.9)

0.34 M sucrose

3 mM CaCl2

2 mM Magnesium acetate

0.1 mM EDTA

Plus 1 mM DTT, 0.5 mM PMSF, 5ug/ml Aprotinin, 5ug/ml Leupeptin

Nuclear lysis buffer 2: 50 mM HEPES (pH 7.9)

10% glycerol

150 mM potassium acetate

1.5 mM MgCl2

0.1% NP-40

Plus 1 mM DTT, 0.5 mM PMSF, 5ug/ml Aprotinin, 5ug/ml Leupeptin

Washing buffer: 20 mM HEPES (pH 7.9)

150 mM KCl

0.5 mM EDTA

0.1% Triton X-100

10% glycerol

Plus 1 mM DTT, 0.5 mM PMSF