Please follow the steps below.
Preparation of enzyme and buffer for use with samples
The papain enzyme (such as the Merck P3125) will be supplied as an aqueous suspension. It should be supplied with a label or datasheet to say how much actual enzyme is dissolved per unit volume — this may vary from batch to batch. We suggest using about 5mg of the active papain. Due to batch-to-batch variation the volume required to do this can therefore vary. You will need to check this datasheet and then calculate what volume of papain suspension will be required to contain 5mg of the enzyme.
The papain suspension will not digest any protein until it is first activated – this is achieved by diluting into a phosphate buffer containing the necessary cofactors. The buffer recipe is provided on p3 of the manual.
Step 1: Prepare the phosphate buffer at the correct pH. The following website may be helpful: phosphate buffer pH 5.8 to 7.4. A 50 ml volume of this will then be set aside; the cofactors and enzyme will be added to this 50ml volume.
Step 2: Add 400 mg sodium acetate
Step 3: Add 200 mg EDTA, disodium salt
Step 4: Add 40 mg cysteine HCl
Step 5: Stir to dissolve all components.
Step 6: Then add your calculated amount of the Papain suspension (see above), making sure you have calculated the volume so that you are adding a total of 5 mg of active enzyme.
Step 7: Stir to dissolve.
This can then be used with your sample.
