Pronase E (Activity ≥ 7000 U/g)

Pronase E (Activity ≥ 7000 U/g) is a mixture of proteolytic enzymes that is obtained from Streptomyces griseus and could digest protein into individual amino acids^[1]. In Vitro: Pronase E significantly increases the amount of most of the amino acids analysed (PE vs C) , especially Ile, His and Thr^[1].

Pronase E Usage Steps:.
1. Preparation of Stock Solution:
(1) Dissolve Pronase E in deionized water to prepare a stock solution at a concentration of 5-20 mg/ml.
Note: If used for DNA or RNA isolation steps, it is recommended to first heat the solution at 56°C for 15 minutes.
(2) Incubate at 37°C for 1 hour. Aliquot the prepared stock solution into single-use samples and store at -20°C; typically stable for one year.
2. DNA Isolation:
Directly add Pronase E to the DNA preparation system. The system should contain 0.5-1% SDS to disrupt DNA-protein interactions.
Note: Use at a concentration of 250-500 μg protein/ml. Incubation conditions: Incubate at 37°C for 1-4 hours.
3. Protein Digestion:
(1) Dissolve approximately 0.2 μM protein in 0.2 ml of 50 mM ammonium bicarbonate buffer (pH 8.0) or phosphate buffer (pH 7.0).
(2) Add 1% (w/w) Pronase E. Incubate at 37°C for 24 hours to perform protein digestion.
In Vivo: Experimental Procedure for Isolating Primary Hematopoietic Stem Cells (HSCs) from Mice Using Pronase E^[2]
1. Animal Anesthesia and Abdominal Exposure:
(1) Anesthetize male or female mice.
(2) Under sterile conditions, expose the liver via abdominal incision.
2. Liver Perfusion Procedure:
(1) Perform liver perfusion through the portal vein using Hanks' Balanced Salt Solution (HBSS) to remove blood and impurities.
(2) Conduct enzymatic perfusion with Pronase E and collagenase to dissociate liver tissue.
3. Liver Extraction and Cell Release:
(1) Carefully extract the liver from the abdominal cavity using sterile forceps, and quickly transfer it to a sterile culture dish.
(2) Gently peel the liver capsule and apply mild mechanical agitation to release cells from the liver tissue.
4. Cell Digestion and Preliminary Centrifugation:
(1) Suspend the released cells in a mixed enzyme solution containing Pronase E, collagenase, and DNase, and digest at 37°C for 20 minutes.
(2) Perform two rounds of low-speed centrifugation (50 g, 3 minutes) to separate liver cells, and collect the cell suspension.
5. HSC Separation and Density Gradient Centrifugation:
(1) Further centrifuge the cell suspension (450 g, 8 minutes) and collect the pellet.
(2) Resuspend the pellet in 18% Nycodenz solution as the basis for density gradient separation.
(3) Construct a density gradient with 12% Nycodenz, 8.2% Nycodenz, and Gey's Balanced Salt Solution (GBSS).
(4) Isolate and purify HSCs from the 8.2% Nycodenz layer through density gradient centrifugation.
Note: This protocol provides standard operating procedures; please adjust and optimize according to specific experimental needs and conditions.