Pronase E (Activity ≥ 4000 U/mg)  (Synonyms: プロナーゼ E (Activity ≥ 4000 U/mg); Pronase (Activity ≥ 4000 U/mg))

Pronase E (Activity ≥ 4000 U/mg) is a mixture of proteolytic enzymes that is obtained from Streptomyces griseus and could digest protein into individual amino acids^[1].

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.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

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.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

9036-06-0

Solid

Off-white to light yellow

[Pronase E (Activity ≥ 4000 U/mg)]

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [1]. José M Bruna, et al. Enhancement of the flavour development of dry fermented sausages by using a protease (Pronase E) and a cell‐free extract of Penicillium camemberti. Journal of the science of food and agriculture. Volume82, Issue5. April 2002. Pages 526-533.

  [2]. Chen QT, et al. HK1 from hepatic stellate cell-derived extracellular vesicles promotes progression of hepatocellular carcinoma. Nat Metab. 2022 Oct;4(10):1306-1321.  [Content Brief]