Protease inhibitors



To prevent degradation of your favorite protein in an extract you may add protease inhibitors. The danger of protein degradation depends very much on the type of extract and amount of protein.
When isolating overexpressed proteins from E. coli under native conditions, you may add PMSF, ortho-Phenanthrolin and Benzamidine. Do not add protease inhibitors when you purify in denaturing conditions. Similarly, use only PMSF when you prepare large-scale extracts from embryos.

You may add a cocktail of the other inhibitors when you are confronted with degradation in certain assays (binding tests) or when you express proteins in retikulocyte lysate. Some of our proteins are rather stable (e. g. Dia) while others (kuk, slam) are instable after solubilisation and require protease inhibitors.

  • PMSF: stock 100 mM in 2-Propanol, use at 1 mM. PMSF has only a limited stability in water (1h). Store at -20°C Inhibits Serin proteases.
  • ortho-Phenanthrolin: stock 100 mM in 2-Propanol, use at 0.25 mM. Chelator of metal ions, inhibits metallo proteases
  • Benzamidin-HCl: do not store in solution, use at 0.25 - 5 mM. Inhibitor of trypsin-like proteases
  • Leupeptin : stock 10 mM, use at 10 µM, (Sigma L2884)
  • Bestatin (Sigma B8385): stock 1 mM, use at 1 µM
  • Pepstatin A (Sigma P5318): stock 1 mM, use at 1 µM
  • E-64 (Sigma E3132): stock 1 mM, use at 1µM
  • Aprotinin A (Sigma A1153): stock 5 mg/ml, use at 5µg/ml

store at -80°C in aliquots


Micro dialysis:


MF Membrane, 0.025m 13 mm, 100 St.
Millipore VSWP01300, 248 €
Anja_Sprunk@millipore.com
Tel.:  06196-494260
Kundennr.:   G245333-B1115834-S1115833


Amidoschwarzfärbung:


Die Amidoschwarzfärbung dient als Schnelltest, um zu testen welche Fraktionen Protein enthalten.
je 1µl der Proteinlösungen werden auf einen Nitrocellulosefilter aufgebracht und an der Luft getrocknet. Nach kurzem Fixieren in Entfärbelösung (1 min) wird der Filter in Amidoschwarzlösung für 5 min gefärbt. Danach wird der Filter in Wasser entfärbt bis der Hintergrund nahezu weiß ist und getrocknet.

Amidoschwarz 10B (für die Mikroskopie), Merck,
0,1% (w/v) in 7% Essigsäure