Table 2.
Examples of lysis buffers that in our hands have yielded successful isolations of different viral proteins with a range of properties.
| Bait | Description & Localization |
Tag | Virus | Cell type |
aOptimized Lysis Buffer | Ref |
|---|---|---|---|---|---|---|
| nsP3 | Non-structural protein 3 (cytoplasm) |
GFP | Sindbis Virus |
Rat2 cells |
1% Triton, 0.5% deoxycholate, 500 mM NaCl, 25 units/mL DNase |
(2) |
| pUL38 | Early phase non-structural protein(dynamic localization in nucleus and cytoplasm) |
ProtA | HCMV | HFF | 1% Triton X-100, 250 mM NaCl, 4 μg/mL DNase, 20 mg/ml phenylmethylsulfony fluoride, 0.4 mg/mL pepstatin A |
(11) |
| pUL99, pUL32 |
Both involved in assembly (cytoplasm) |
GFP | HCMV | HFF | 1 µM ZnCl2, 1 µM CaCl2, 1% Triton, 250 mM NaCl |
(5) |
| pUL83 | Tegument protein; (nuclear and cytoplasmic) |
GFP ProtA |
HCMV | HFF | 1% Triton X-100, 250 mM NaCl, 4 μg/mL DNase, 20 mg/ml phenylmethylsulfony fluoride , 0.4 mg/mL pepstatin A |
(6) |
| pUL27 | Early phase protein (nuclear) | 3xFLAG | HCMV | HFF | 1% Triton X-100, 250 mM NaCl, 4 μg/mL DNase |
(9) |
| nsP4 | Non-structural RNA- dependent RNA polymerase (cytoplasmic) |
3xFLAG | Sindbis Virus |
Rat2 cells |
1% Triton X-100, 0.5% deoxycholate, 500 mM NaCl, 25 U DNase and RNase/mL |
(8) |
| NS1 | Non-structural glycoprotein (cell surface and secreted) |
No tag | West Nile Virus |
BHK2 1-15 |
1 µM ZnCl2, 1 µM CaCl2, 1% Triton, 0.5% sodium deoxycholate, 0.3% sodium N-lauroylsarcosine, 0.1 M NaCl, and phosphatase inhibitor cocktail |
(10) |
a
Alllysis buffers contained 20 mM K-HEPES pH 7.4, 110 mM KOAc, 2 mM MgCl2, 0.1% Tween 20, protease inhibitor mixture in addition to optimized lysis buffer components listed.