Protease inhibitor cocktail

From Sigma 3-aminoethylcarbazole (AEC) acrylamide/bis-acrylamide (30%) 37.5 1 amino acids alumina bentonite benzamidine bovine fiver tRNA bovine serum albumin (BSA) creatine phosphate (CP) diethyl pyrocarbonate (DEPC) dithiothreitol (DTT) Escherichia coli MRE600 tRNA pyrophosphatase (Ppase) Ca++ salt of folinic acid, (5-formyl THF) IIHPHS K salt of phospho-enol pyruvic acid, (PEP) creatine phospho kinase (CPK) protease inhibitor cocktail for fungal and yeast extracts phenylmethylsulfonyl fluoride (PMSF) spermidine trihydrochloride Tween 20. 

Because PMSE fails to inactivate acetylcholinesterase, this reagent is much less toxic than diisopropylfluoro-phosphate, and is also recommended as an alternative to the neurotoxic fluorophosphates and fluorophospho-nates. PMSE is freshly prepared as a 1-3 mM solution in water (higher concentrations will precipitate spontaneously). A better procedure is to first prepare a 20 mM PMSE solution in 2-propanol or dioxane this solution can then be added to the biological fluid with vortex mixing to achieve a 1-3 mM final concentration as a homogeneous solution. One should confirm that the alcohol or dioxane has little or no undesirable effect on enzymes or proteins of interest. See Chymotrypsin Protease Inhibitor Cocktails ... 

Mammalian Cell Protease Inhibitor CocktaiL These should contain AEBSF, pepstatin A, E-64, bestatin, leupeptin, and aprotinin. (Metal chelators can be added to suppress the activity of calcium ion-dependent proteases such as calpain. Again, one must determine whether the protein or enzyme being purified does not require a divalent metal cofactor for stabihty or activity.)... 

Protease inhibitor cocktail (for histidine-tagged proteins) (Sigma-Aldrich, St. Louis, MO). 

Pellet the cells by centrifugation at 4° C and freeze at-70°C or in a dry ice/ethanol bath. Prepare fresh lysis buffer by adding 1 mg/mL lysozyme, 2.5 U/mL Benzonase nuclease, 2 m. / MgCk 2 pL/mL protease inhibitor cocktail, and 1 mA/PMSF. Resuspend the frozen pellets in 500 pL fresh lysis buffer and shake on a plate shaker at 4°C for 30 min to lyse the cells. 

Thaw bacterial pellets in 24-well blocks at room temperature. While waiting, add protease inhibitors, lysozyme, and Benzonase to the chilled lysis buffer 1 mg/mL lysozyme, 10 units Benzonase/mL, 2 pL/niL protease inhibitor cocktail (no ethyl-enediamene tetraacetic acid [EDTA]), and 1 mA/PMSF. Keep on ice. 

Harvest cells by centrifuging at 5000for 15 min at 4 °C. Remove the supernatant and resuspend the cell pellet in 50 ml oflysis buffer + 500 p of bacterial protease inhibitor cocktail (Sigma). At this point, the cells can be frozen and stored at — 80 °C. 

Different extraction techniques have been used for the characterization of the artichoke s proteome by CPLL. The initial extraction buffer contained 50 mM Tris-HCl (pH 7.4), 50 mM sodium chloride, 2% (m/v) CHAPS, 1% (m/v) sodium dodecyl sulfate, and 25 mM dithiothreitol. Protease inhibitor cocktails were added to the extraction buffers to prevent the action of protease. The first extract was then diluted 1 10 (v/v) with the same buffer without sodium dodecyl sulfate to facilitate the protein capture. The extract was separated into four equal aliquots each of them was titrated to different pH values (4.0, 7.2, and 9.3). The pH of the fourth aliquot was reduced to 2.2 with addition of 0.1% TFA and formic acid. Then individually, each aliquot was added with 100 pL of CPLL beads overnight at room temperature under gentle shaking. 

Proteins were then dissolved in 20 mL of 0.125 M Tris-HCl, pH 7.4 containing 1% (m/v) CHAPS and the protease inhibitor cocktail, and treated with CPLL. With several variations from the standard treatments such as other complementary extractions from seeds, the use of urea-containing extraction buffer, and an additional peptide ligand library, the total number of detected gene products was quite large. Two hundred and thirty one unique proteins were found in the pulp (vs. 56 described previously) and 61 in the seeds (vs. only four reported by the literature). In the latter case, the presence of seed storage proteins, oleosins, and histones were detected in the pulp, a thaumatin-like protein, an allergenic protein also named Ole el3, was confirmed. 

Homogenization buffer, pH 7.4. Concentrations given below indicate the final concentrations. The concentrations of stock solutions that can be used are indicated. 50 mM HEPES (stock solution 1M), 1 mM EDTA 0.5 M (stock solution pH 8.0), 10 mM MgCl2 (stock solution 1M), Protease inhibitor cocktail tablets (see Note 2), 2 iM Pepstatin A (stock solution 1 mM in 100% DMSO), 1 mM phenylmethyl-sulfonyl fluoride (PMSF) (stock solution 50 mM in 100% alcohol) (see Note 2). 

The protease inhibitor cocktail tablets Complete from Roche Molecular Biochemicals, Rotkreuz, Switzerland, Cat. No. 1 697 498. PMSF Phenylmethyl-sulfonyl fluoride, Sigma Cat. No. P-7626. 

Lysis buffer 0.5% NP40 (Sigma), 25 mMTris-HCl, pH 7.5, 5 mMEDTA, 150 mMNaCl, protease inhibitor cocktail (Roche) (see Note 3)... 

Hyptonic buffer 5 mMTris-HCl, pH 7.5,5 mMEDTA, protease inhibitor cocktail... 

A mixture of protease inhibitors, which is used to preserve protein integrity during the processing of samples for subsequent analysis. The term cocktail refers to a mixture of components. A protease inhibitor cocktail is composed of a broad spectrum of protease inhibitors and intends to inhibit the diverse proteolytic enzymes found in tissue extracts and biological fluids. See Pringle, J.R., Methods for avoiding proteolytic artifacts in studies with enzymes and other proteins from yeasts. Methods Cell Biol. 12, 149-184, 1975 Dmbin, D.G., Miller, K.G., and Botstein, D., Yeast... 

Characteristics of Selected Protease Inhibitors, Which Can Be Used in Protease Inhibitor Cocktails... 

The protease inhibitor cocktails referred to herein are not to be confused with the protease inhibitor cocktails that are used for therapy for patients who have Acquired Immune Deficiency Syndrome (AIDS). 

Cell pellets from 4L of baculovirus infected insect cells were resuspended into 25mM Tris-HCL, pH8.0, 5mM DTT, 250mM sucrose and protease inhibitor cocktail tablets (Boehringer Mannheim). Cells were homogenized and centrifuged at 38,000 X g for 20 min. The supernatant was ultracentrifuged at 100,000 X g for 2hrs, filtered, and loaded over a 21.5mm ID X 15 cm DEAF column... 

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