Protease Inhibition using Protease Inhibitor Mix

Extracted from 2-D Electrophoresis Principles and Methods
GE Healthcare, 2004

Protease Inhibitor Mix from GE Healthcare contains an optimized concentration of competitive and noncompetitive protease inhibitors that effectively inhibit serine, cysteine, metalloproteases, and calpain proteases. The kit is suitable for the protection of proteins during purification from animal tissues, plant tissues, yeast, and bacteria.

Protocol: Protease Inhibitor Mix

Reagents supplied

Protease Inhibitor Mix (100× solution), 1 ml.

Required but not provided

Microcentrifuge, vortex mixer, extraction solution.

Preliminary notes

Protease Inhibitor Mix is provided free of EDTA as some proteins require divalent cations such as Ca2+, Mg2+, or Mn2+ for their biological activity. In such circumstances, the presence of EDTA may be detrimental to sample protein activity.

Samples can be extracted into 8 M urea and 4% CHAPS, or into 7 M urea, 2 M thiourea, and 4% CHAPS (see solutions A and B in appendix I). Alternative nonionic detergents or protease inhibitors can be added during extraction. Carrier ampholytes (Pharmalytes, Ampholines, or IPG Buffers) can be added at concentrations up to 2% for standard protocols but should not be added during protein extraction for labeling in 2-D DIGE.

1. Allow the solution to warm to room temperature.
2. Vortex briefly before using, as the solution is in suspension form.
3. Dilute Protease Inhibitor Mix 1:100 (10 μl/ml) in an appropriate volume of extraction buffer or extract.

Further options

• If a higher potency of protease inhibition is required, add Protease Inhibitor Mix at a concentration of 20–30 μl/ml to give a 2–3× final concentration.
• For the inhibition of metalloproteases, add EDTA directly in an appropriate volume of extraction buffer or extract to give a final concentration of 5 mM EDTA in the reaction.
  
  EDTA must not be added if the solution is to be used in conjunction with Nuclease Mix, because EDTA acts as a nuclease inhibitor.

A. Sample preparation solution (with urea) for 2-D electrophoresis

[8 M urea, 4% CHAPS, 2% Pharmalyte or IPG buffer (carrier ampholytes), 40 mM DTT, 25 ml]

	Final concentration	Amount
Urea (FW 60.06)	8 M*	12 g
CHAPS†	4% (w/v)	1.0 g
Pharmalyte‡ or IPG Buffer§	2% (v/v)	500 μl
DTT (FW 154.2)	40 mM	154 mg
Double-distilled water	—	to 25 ml (16 ml required)

* If necessary, the concentration of urea can be increased to 9 or 9.8 M.
† Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).
‡ Carrier ampholytes (Pharmalyte or IPG buffer) and DTT should be excluded from the sample extraction solution if the samples are to be labeled using 2-D DIGE. See Ettan DIGE User Manual for details.
§ Use IPG Buffer in the pH range corresponding to the pH range of the IEF separation to be performed, or Pharmalyte in a pH range approximating the pH range of the IEF separation to be performed.

Store in 2.5-ml aliquots at -20 °C.

Note: Protease inhibitors may be added if necessary.

B. Sample preparation solution (with urea and thiourea) for 2-D electrophoresis

[7 M urea, 2 M thiourea, 4% CHAPS, 2% Pharmalyte or IPG Buffer (carrier ampholytes), 40 mM DTT, 25 ml]

	Final concentration	Amount
Urea (FW 60.06)	7 M	10.5 g
Thiourea (FW 76.12)	2 M	3.8 g
CHAPS*	4% (w/v)	1.0 g
Pharmalyte† or IPG Buffer‡	2% (v/v)	500 μl
DTT (FW 154.2)	40 mM	154 mg
Double-distilled water	—	to 25 ml (13.5 ml required)

* Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).
† Carrier ampholytes (Pharmalyte or IPG buffer) should be excluded from the sample extraction solution if the samples are to be labeled using 2-D DIGE.
‡ Use IPG Buffer in the pH range corresponding to the pH range of the IEF separation to be performed, or Pharmalyte in a pH range approximating the pH range of the IEF separation to be performed.

Store in 2.5-ml aliquots at -20 °C