Pronase E (Activity ≥ 7000 U/g)

Pronase E Usage Instructions ^[1]:

Pronase E is highly stable within the pH range of 5.0-9.0, with maximum enzyme activity at pH 8.8. Compatible with many isolation buffer systems. Pronase E has broad substrate specificity; can degrade proteins to individual amino acids. Complete inactivation can be achieved by heating to 80 ℃ or higher for 15-20 minutes.

Unit Definition: Under conditions of 37 ℃ and pH 7.5, the enzyme quantity required to produce a color change equivalent to 1 µM of tyrosine (181 g of casein) per minute, as measured by the Folin-Phenol reagent method, is defined as one activity unit.

This product is widely used. Specific usage methods and working concentrations should be adjusted based on experimental experience or reference literature.

Preparation of Stock Solution: Dissolve in deionized water to prepare a stock solution with a concentration of 5-20 mg/ml. If used in DNA or RNA isolation steps, it is recommended to heat the solution at 56℃ for 15 minutes and then incubate at 37℃ for 1 hour. Divide the stock solution into single-use aliquots and store at -20℃; typically stable for one year.

DNA Isolation: Directly add to the DNA preparation system (containing 0.5-1% SDS to disrupt DNA-protein interactions). The typical working concentration range is 250-500 μg protein/ml, with incubation at 37℃ for 1-4 hours.

Protein Digestion: Dissolve approximately 0.2 µM of protein in 0.2 ml of 50 mM ammonium bicarbonate buffer, pH 8.0 (or phosphate buffer, pH 7.0). Then, add Pronase E at 1% (w/w) and incubate at 37°C for 24 hours.

Protocol for Isolation of primary hematopoietic stem cells（HSCs) in mice by  Pronase E ^[2]:

1. Male or female mice were anesthetized, and abdominal cavities were opened.
2. The liver was perfused with Hanks' Buffered Salt Solution (HBSS) through portal veins, followed with Pronase E and collagenase perfusion.
3. The liver was then gently removed with sterile forceps and transferred into sterile dishes.
4. The hepatic capsule was teared apart, and the liver was gently shaken to release cells.
5. The cells were suspended in solution containing Pronase E, collagenase and DNase, then digested at 37℃ for 20 min.
6. Hepatocytes were isolated using two rounds of centrifugation at 50g for 3 min.
7. The supernatant was further centrifuged to obtain HSCs at 450g for 8 min, then precipitates were suspended with 18% Nycodenz solution to prepare density gradient centrifugation with 12% Nycodenz, 8.2% Nycodenz and Gey's Balanced Salt Solution (GBSS).
8. Eventually, cells were purified from the layer of 8.2% Nycodenz to obtain HSCs.

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1]. Pronase E significantly increases the amount of most of the amino acids analysed ( PE vs C ) , especially Ile, His and Thr

[2].  Chen QT, Zhang ZY, et,al. HK1 from hepatic stellate cell-derived extracellular vesicles promotes progression of hepatocellular carcinoma. Nat Metab. 2022 Oct;4(10):1306-1321. doi: 10.1038/s42255-022-00642-5. Epub 2022 Oct 3. PMID: 36192599; PMCID: PMC9584821.