Enzyme Digestion Methods

Enzyme Digestion

Ready to use digestion enzymes: - These may be ‘generic’ unmasking enzyme- formulations to be appropriate for a range of digestions required and are convenient to use, both for researchers doing small numbers of slides where excess enzyme working solutions would normally be wasted, or in high throughput labs where consistency of reagents is paramount and there is little time for daily preparation of solutions.

Fast Enzyme

A Ready to use enzyme solution that works for most enzyme detection methods.  Although it works at room temperature, rather than requiring to be heated to 37°C, be mindful that the application temperature should be consistent, or timings may be variable.  If optimization has been done with reagent at room temperature, then reagent taken straight from the fridge may not have complete digestion with the same timing.

User prepared single enzyme reagents:-

 Protease

Protease XXIV, bacterial (EC 3.4.21.62) 7-14 U/mg

PBS pH7.2-7.4 - pre warmed to 37°C

Make a 0.05-0.5% solution of protease in PBS. Incubate for optimised time at 37°C. Rinse well with tap water before continuing the method.

Trypsin enzyme digestion solutions

Trypsin – approx. 435USP units/mg

Calcium Chloride, 0.1% in water, 0.005M Tris/HCl or Tris HCl-buffered 0.9% sodium chloride (TBS) pH 7.6-8.0

0.1M (0.4% sodium hydroxide (aq)

Dissolve trypsin in warmed calcium chloride (37°C) to make a 0.1% solution. Quickly pH to 7.8 with sodium hydroxide to maintain temperature. The trypsin may not fully dissolve until the solution reaches near pH 7.8.

Incubation time for trypsin digestion are normally 10-20 minutes. Rinse well with tap water before continuing the method.

Trypsin can also be prepared as a stock solution:-

Dissolve 4g Trypsin (type II – Porcine pancreas) in 100ml dH₂O – aliquot as 5ml & freeze until required

Dissolve 4g Calcium chloride in 100ml dH₂0 - aliquot as 5ml & freeze until required

To use; thaw 1 vial each Trypsin & Calcium chloride, dissolve in 190ml dH₂O, pH to 7.8 with 1N NaOH and warm to 37°C. Optimise in 5 minute increments between 10-30 minutes. This will depend on fixation times & per antigen.

Trypsin Kit

Pepsin

Pepsin (porcine stomach EC 3.4.23.1) 1:2500, 600-1000u/mg      

Hydrochloric acid 0.01 M at 37°C

Make a 0.4% solution of pepsin in HCl. Incubate for optimised time at 37°C. Rinse well with tap water before continuing the method.

Pepsin Solution

Pronase

Incubate 25-37°C for 30 minutes

Pronase

Proteinase K

Incubate 25-37°C for 5 minutes

Neuraminidase

Neuraminidase from Clostridium perfringens (EC 3.2.1.18) 0.5-6 U/mg

0.1M sodium acetate/ acetic acid buffer pH 5.5, stock solution pre warmed to 37°C

0.1% aqueous calcium chloride

Make a solution on 1U/ml in acetate buffer and freeze in 100µl aliquots (0.1U). To use - defrost an aliquot & add to 800µl warm acetate buffer and 100µl calcium chloride solution. Apply drops to sections for about 10 minutes at 37°C. Rinse well with tap water before continuing the method.