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Pefabloc® SC PLUS Protocol

Product No. 11873601001

Protocol

Binding to Proteins

Pefabloc SC (like PMSF) belongs to the sulfonyl-type serine protease inhibitors. These inhibitors are known to covalently bind proteins when used at high concentrations, or during extended incubation times under alkaline conditions. This interaction adversely affects the tyrosine and lysine residues of a protein, as well as the free amino terminus. To avoid this covalent modification, Roche introduced the Pefabloc SC PLUS set, which combines the protease inhibitor Pefabloc SC with a especially formulated Pefabloc SC protector (PSC protector).

Application in Cell Culture

No effects on viability of cell culture cells are found at a Pefabloc SC concentration up to 0.25 mM. Concentrations from 1 to 4 mM result in a reduction of cell viability by approx. 60%. Cytotoxicity was measured using a standard protocol published (MTT assay: Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 55-63).

For inhibition of proteases, a Pefabloc SC concentration between 1 and 5 mM is required. To effectively inhibit proteases in cell culture applications there is also a reduction in cell viability of about 60%. Some publications report that Pefabloc SC is cell permeable, showing intracellular effects at even non-toxic concentrations. Pefabloc SC is one of the most suitable protease inhibitors for cell culture. An alternative serine protease inhibitor could be aprotinin, but aprotinin is not expected to enter into cells.

A further reference for use of serine protease inhibitors in cell culture is Mintz G.R. 1993. An irreversible serine protease inhibitor. Biopharm 6, 34 38.

Inhibition of Proteinase K

Normal working concentrations / starting concentration for Pefabloc SC is between 0.1 - 1.0 mg/ml (0.4 - 4 mM). For inactivation of proteinase K, a TE buffer containing 5 mM Pefabloc SC has been used (4 h, +37 °C, pH 7). No proteinase K activity was detected after this treatment. The extent of proteinase K inhibition will depend on the particular assay conditions. Other experiments with a Tris-HCl buffer, pH 7.8 did not work.

Please note that the stability of Pefabloc SC is affected both by pH and temperature. Pefabloc SC undergoes hydrolysis at pH 7 and above, and the rate is increased at higher temperatures. At pH 7 and +37 °C, Pefabloc SC is hydrolyzed by 50% in 6 h. Under the same temperature, at pH 7.5, the half life of Pefabloc SC is 2 h. For further information on this product, please refer to the package insert of Pefabloc SC.

Materials

     

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