The most critical step in protein-related assays is the extraction of proteins. In the process of extraction, we often add protease inhibitors to prevent protein degradation. In addition, phosphatase inhibitors are also essential in the study of phosphorylated proteins.
This article summarizes the commonly used protease inhibitors PMSF, Leupeptin leupeptin, Aprotinin aprotinin, Pepstatin pepsin inhibitor, EDTA-Na2, etc., as well as phosphatase inhibitor NaF sodium fluoride, Na3VO4 sodium citrate, Beta-glycerophosphate Solution preparation of sodium glycerophosphate, Na2P2O4 sodium pyrophosphate, etc., concentration of stock solution and working solution, and storage conditions.
First, protease inhibitors
PMSF:
Characteristics: Serine protease inhibitors such as chymotrypsin, trypsin and thrombin also inhibit cysteine ​​proteases such as papain.
Solubility: soluble in isopropanol, ethanol, methanol and propylene glycol >10 mg/ml. Unstable in aqueous solution. Stable at 100 ° isopropanol for at least 9 months at 25 °C.
Molecular weight: 174.2
Use: storage concentration 200mM, working concentration 1mM
Leupeptin
Characteristics: inhibition of serine and cysteine ​​proteases such as trypsin, papain, plasmin and cathepsin B.
Solubility: Highly soluble in water (1 mg/ml). Stable at 4 ° C for one week, divided into small portions, frozen at -20 ° C for at least 6 months.
Molecular weight: C20H38N6O4 × 1/2 H2SO4: 475.6 C20H38N6O4 x 1/2 H2SO4 × H2O: 493.6
Use: Store at a concentration of 1 mg/ml and a working concentration of 0.5 ug/ml (1 mM).
Aprotinin aprotinin
Characteristics: Serine protease inhibitor, inhibits the high activity of plasmin, kallikrein, trypsin, chymotrypsin. Does not inhibit thrombin or factor X.
Solubility: Soluble in water (10 mg/ml) or buffer (eg 0.1 M tris, pH 8.0). The solution with a pH of about 7~8 can be stored at 4°C for 1 week, and stored at -20°C for at least 6 months. Avoid repeated freezing and thawing, alkaline environment with pH>12.8 can make it inactivate.
Molecular weight: 6,512
Use: storage concentration 1mg/ml, working concentration 0.06~2.0 ug/ml (0.01~0.3 uM).
Pepstatin pepsin inhibitor
Characteristics: inhibition of aspartic acid (acid) proteases such as pepsin, renin, cathepsin D, chymosin, many microbial acid proteases
Solubility: soluble in methanol about 1mg/ml; soluble in ethanol, dissolved up to 1 mg/ml overnight; solubility in 6 equivalents of acetic acid is 300ug/ml. Stable at 4 ° C for one week, stored at -20 ° C for 1 month.
Molecular weight: 685.9
Use: Store at a concentration of 1 mg/ml at a concentration of 0.7 μg/ml (1 μM).
EDTA-Na2
Characteristics: metalloproteinase inhibitor
Solubility: soluble in water to 0.5M, stable at 4°C for at least 6 months at pH 8-9
Molecular weight: 372.24
Use: Working concentration 0.2~0.5 mg/ml (0.5~1.3 mM), no need to use the current match, add when the pH value of the solution is adjusted to 8~9.
Second, phosphatase inhibitors
NaF sodium fluoride
Solubility: soluble in water
Molecular weight: 41.99
Use: 5M stock solution, working concentration 10~20mM.
Na3VO4 sodium orthosilicate
Molecular weight: 183.91
Solubility: Soluble in water, we bought sodium citrate. Sodium citrate needs to be treated to become activated sodium citrate, and activated sodium citrate has the effect of inhibiting dephosphorylation. The process of sodium decanoate becoming activated sodium citrate is: 100 mM sodium citrate activated storage solution
(1). Dissolve 0.183 g of sodium decanoate in 10 ml of double distilled water and adjust the pH to 10 (yellow color) with acid.
(2). Cook until colorless
(3). Room temperature cooling
(4). Reset the pH to 10
(5). Repeat (1) (2) (3) (4) until the solution remains colorless, and the pH is stable at 10, dispense 100 ul / tube, add one tube per 10 ml lysate.
Use: stock solution 100 mM, working concentration 1 mM, stored at -20 degrees.
Beta-glycerophosphate sodium glycerophosphate
Solubility: soluble in water
Molecular weight: 306.11
Use: stock solution 100 mM, working concentration 25 mM.
Na2P2O4 sodium pyrophosphate
Solubility: soluble in water
Molecular weight: 265.9
Use: stock solution 100 mM, working concentration 1-2 mM.
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