Western Blot樣品製備–蛋白酶抑製劑
在Western Blot樣品製備步驟中，當樣品經裂解液反應後，就會開始蛋白水解、去磷酸化和變性等等反應。因此若能將樣品持續保存在冰上(4°C以下)，並在裂解緩衝液中加入適當的新鮮抑製劑，則可以顯著減緩蛋白被降解。
以下為常見的抑制劑種類，可自行搭配混和抑制劑
|
Inhibitor |
Protease/phosphatase inhibited |
Final concentration in lysis buffer |
Stock (store at -20°C) |
|
Trypsin, chymotrypsin, plasmin |
2 µg/mL |
Dilute in water, 10 mg/mL. Do not re-use thawed aliquots. |
|
|
Lysosomal |
5–10 µg/mL |
Dilute in water. Do not re-use thawed aliquots. |
|
|
Aspartic proteases |
1 µg/mL |
Dilute in methanol, 1 mM. |
|
|
Serine, cysteine proteases |
1 mM |
Dilute in ethanol. You can re-use the same aliquot. |
|
|
EDTA |
Metalloproteases that require Mg2+ and Mn2+ |
5 mM |
Dilute in dH20, 0.5 M. Adjust pH to 8.0. |
|
EGTA |
Metalloproteases that require Ca2+ |
1 mM |
Dilute in dH20, 0.5 M. Adjust pH to 8.0 |
|
Sodium fluoride |
Serine/threonine phosphatases |
5–10 mM |
Dilute in water. Do not re-use once defrosted. |
|
Tyrosine phosphatases |
1 mM |
Dilute in water. Do not re-use once defrosted. |
Abcam也提供Inhibitor Cocktail(含EDTA/不含EDTA)版本可供選擇
|
Product Name |
Cat# |
|
Protease Inhibitor Cocktail |
|
|
Protease Inhibitor Cocktail (EDTA-free) |
|
|
Protease and Phosphatase Inhibitor Cocktail |
|
|
Protease and Phosphatase Inhibitor Cocktail (EDTA-free) |
