Protease inhibitor
What is the function of protease inhibitors?
The purification and extraction of proteins are often influenced by the hydrolytic enzymes present in them. During the process of protein extraction, cells break down and release proteases. When these enzymes come into contact with the protein to be extracted, they react under suitable conditions, leading to protein breakdown. Therefore, in the process of protein extraction, it is necessary to add protease inhibitors to prevent protein hydrolysis and maintain protein integrity.
What is the principle of action of protease inhibitors?
Protease inhibitor broadly refers to a substance that binds to some functional groups on the active center of a protease molecule, causing a decrease or even disappearance of protease activity, but not causing denaturation of the enzyme protein.
What are the conditions for using protease inhibitors?
1. Because various proteases have the same sensitivity to different proteins, it is necessary to adjust the concentration of each protease.
2. Due to the extremely low solubility of protease inhibitors in liquids, it should be noted that when adding protease inhibitors to the buffer, they should be thoroughly mixed to reduce the precipitation of protease inhibitors.
What are the commonly used protease inhibitors?
1. Phenylmethylsulfonyl fluoride (PMSF)
① Inhibit serine proteases (such as chymotrypsin, trypsin, thrombin) and thiol proteases (such as papain), but do not inhibit metalloproteinases and most cysteine proteases and aspartic proteases; ② Dissolve in isopropanol, ethanol, methanol, and 1,2-propanediol. Unstable in aqueous solution, stable at 259 ℃ in 100% isopropanol; ③ Can be stored for one year at room temperature; ④ Working concentration: 0 1 ~ 1. 0mmol/L;⑤ Unstable in aqueous solution, fresh PMSF must be added in each separation and purification step. 2. Sodium ethylenediaminetetraacetate (EDTA-Na2) ① inhibits metalloproteases; ② Dissolve in water with a pH of 8-9, with a solution concentration of approximately 0.5mol/L The solution can be stable for more than 6 months at 4 ℃; ④ Working concentration: 0.2~1.5mmol/L; ⑤ NaOH needs to be added to adjust the pH of the solution, otherwise EDTA will not dissolve.
3. Pepsin inhibitor
① Inhibit acidic proteases (such as pepsin), angiotensinogenase, protease L, and rennet; ② 1mg/mL soluble in methanol, insoluble in water; ③ The storage solution is stable within one week at 4 ℃ and can remain stable for 6 months at -209 ℃ Working concentration: 0 7-1 μ mol/L.
4. Luminescent protease inhibitory peptide
① Inhibit serine and thiol proteases, such as papain, plasma enzymes, and protease B; ② 10mg/mL dissolved in water; ③ Stable for one week at 4C storage solution, and stable for 6 months at -20 ° C; ④ Working concentration: 0 5mg/mL.
5. Trypsin inhibitor
① Inhibit serine proteases (such as plasma enzymes), angiotensin, trypsin, and chymotrypsin; ② 10mg/mL dissolved in water, pH 7-8, deactivated at pH>12.8; ③ Stable for one week at 4 ° C and for six months at -209 ° C to avoid repeated freeze-thaw cycles Working concentration: 0 01~0.3 μ mol/L
6. Gastric suppressant peptide
① Inhibit aspartic proteases (such as pepsin), renin, protease D, rennet, and many acidic proteases of microorganisms; ② Dissolved in methanol, about 1mg/mL, also soluble in ethanol or 6mol/L acetic acid (about 300ug/mL), insoluble in water, stable for at least one week at 4 ° C; ③ Working concentration: 0 7 μ g/mL.
7. Cystatine inhibitor
① Inhibit cysteine proteases such as papain, ficin, protease B, and dipeptidyl peptidase; ② Dissolve 500 μ g of cysteine protease inhibitor in distilled water containing 20% glycerol, freeze at -20 ° C, and stabilize for two months; ③ Working concentration: 250 μ g/mL.
phosphatase inhibitors
What is the function of protease inhibitors?
During cell or tissue lysis, a large amount of endogenous protein phosphatase is released, which catalyzes the dephosphorylation of phosphorylated proteins in various ways, thereby affecting subsequent protein detection. Therefore, adding an appropriate amount of exogenous phosphatase inhibitor to the extract is beneficial for inhibiting the dephosphorylation of phosphorylated proteins and maintaining the phosphorylation state of proteins in subsequent experiments such as Western blotting, immunoprecipitation detection of phosphorylated proteins, and protein kinase activity determination.
What is the principle of action of protease inhibitors?
In cellular signal transduction, target proteins are phosphorylated by protein kinases that can transfer phosphate groups to specific sites. These phosphate groups are removed by endogenous protein phosphatases during cell lysis. Phosphatase inhibitors help maintain the activity and state of phosphoproteins during cell lysis and protein extraction processes.
What are the commonly used protease inhibitors?
Common phosphatases include protein tyrosine phosphatase, alkaline phosphatase, and acid phosphatase. The commonly used phosphatase inhibitors are as follows:
1. Sodium fluoride (sodium μ m fluoride): a reversible inhibitor of acid phosphatase;
2. Sodium pyrophosphate: an irreversible inhibitor of serine/threonine phosphatase;
3. β – glycerophosphate: a reversible inhibitor of serine/threonine phosphatase;
4. Sodium orthovanadate: reversible inhibitor of alkaline phosphatase and tyrosine phosphatase:
5. Sodium molybdate (sodium μ m molybdate): an irreversible inhibitor of acid phosphatase;
6. Sodium tartrate dihydrate: a reversible inhibitor of acid phosphatase;
7. Imidazole: a reversible inhibitor of alkaline phosphatase.