Table 3.
Examples of lysis buffer conditions that we previously used to isolate mammalian host proteins from both cultured cells and tissue.
| Bait | Description & Localization |
Tag | Cell type | Optimized Lysis Buffer | Ref |
|---|---|---|---|---|---|
| Nup37 Nup43 |
Member of nuclear pore complex and subunit of Nup107-160 subcomplex (nuclear membrane) |
GFP | HeLa | 0.5% Triton, 200 mM NaCl, 20 mg/mL PMSF, 0.4 mg/mL pepstatin A |
(1) |
| GluRδ2 | postsynaptic densities; (cerebellar excitatory synapses) |
GFP | Mouse tissue |
b 10 mM HEPES, pH 7.4, 2 mM CaCl2 , 132 mM NaCl, 3 mM KCl, 2 mM MgSO4, 1.2 mM NaH2PO4, 0.5% Triton X-100, 1/100 (v/v/) protease inhibitor cocktail |
(25) |
| HDAC1 | Histone deacetylase 1 (nucleus) |
GFP | HFF | 1µM ZnCl2, 1 µM CaCl2, 0.5% Triton X- 100, 250 mM NaCl |
(12) |
| H3 | Histone 3 isoforms (nucleus) | YFP | mouse ES cells |
0.5% Triton, 300 mM NaCl | (24) |
| HDAC5 | Histone deacetylase 5 (nucleus and cytoplasm) |
GFP | HEK293 | 1 μM ZnCl2, 1 μM CaCl2, 0.5% Triton X- 100, 250 mM NaCl, 4 μg/mL DNase, and phosphatase inhibitor cocktails |
(13) |
| SIRT7 | Sirtuin 7 (nucleoli) | GFP | HEK293 | 1 μm ZnCl2, 1 μm CaCl2, 0.5% Triton X- 100, 250 mm NaCl, 4 μg/ml DNase, and phosphatase inhibitor cocktails |
(14) |
| IFI16 | Interferon inducible protein 16 (nucleus and cytoplasm) |
No tag | CEM T | 1 μM ZnCl2, 1 μM CaCl2, 0.6% Triton X- 100, 200mM NaCl, 10 μg/mL DNase I, phosphatase inhibitor cocktail |
(16) |
a
Alllysis buffers contained 20 mM K-HEPES, pH 7.4, 110 mM KOAc, 2 mM MgCl2, 0.1% Tween 20, 1/100 protease inhibitor mixture in addition to optimized lysis buffer components listed.
b
This buffer is the complete buffer composition without core components listed above.