formalin (35% formaldehyde), 2% (w/v) sodium carbonate (Na2CO3); (e) Stop solution: 5% (v/v) glacial acetic acid. All chemicals supplied by Sigma-Aldrich (St. Louis, MO) and are made fresh in ultrapure water.
Nalgene Staining box, 22.5 x 22.5 x 5 cm (Sigma-Aldrich).
1. pRevTre retroviral response vector and pRevTet-On retroviral expression vector (Clontech, Palo Alto, CA) (see Note 3).
2. Doxycyclin (Clontech, Palo Alto, CA) stock solution. Dissolve at 1 mg/mL in water and filter sterilize. Store at 4°C in the dark, and use within 4 wk. Aliquots can be frozen at -20°C for long-term storage. Fresh doxycyclin should be added to the media for each experiment.
3. MNT buffer: 20 mM2-(N-morpholino)ethanesulfonic acid (MES); 100 mM NaCl; 30 mM Tris-HCl, pH 7.5; 1 mM EDTA; 1 mM PMSF; 0.5% Triton X-100. Prepare in small volumes and store at 4°C, stable for 1 mo.
4. Agarose coupled M2 anti-FLAG antibody (Sigma-Aldrich) should be stored at -20°C. Prepare by centrifuging required amount of agarose beads at 5,000g for 30 s and resuspend in 1 mL of MNT buffer containing 0.1% SDS (MNT-S). Wash 2X with MNT-S and resuspend as a 50% slurry in MNT buffer, make fresh as required. FLAG peptide stock (400 pg/mL): resuspend 4 mg of lyophilized FLAG peptide in 10 mL of MNT-S buffer. Store aliquots at -20°C and avoid freeze/thawing.
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