EnPresso® B Defined Nitrogen-free 500 Protocol

Product No. B21250


EnPresso® B Defined Nitrogen-free 500 is a pre-sterilized, chemically-defined growth system designed to increase the yield of 15N-labelled proteins.

EnPresso growth systems provide optimal conditions for growth, metabolism and protein expression in microbial cultures. Protein yields are increased by enabling cultures to reach far higher cell densities than those achieved using conventional media. By controlling growth rate and metabolism, a greater proportion of expressed protein can be correctly folded to improve solubility, minimize the risk of inclusion body formation, and ensure functionality of the final product.

EnPresso growth systems maintain pH, provide adequate minerals, vitamins and trace elements to support growth and use proprietary EnBase® technology to ensure a constant slow release of glucose from a polysaccharide substrate.

EnPresso® B Defined Nitrogen-free 500

EnPresso® B Defined Nitrogen-free 500 (B21251)

Tablets, silver bag: 2 bags
Each bag contains 20 tablets composed of minerals, vitamins, trace elements and polysaccharide substrate
Reagent A (3000 U/L)
5 mL
Glucose-releasing agent
Format Each bag contains sufficient components for a 500 mL culture. Tablets and reagent are manufactured using standard aseptic techniques and filtration or gamma irradiation to ensure sterility.
Storage Store unopened kit contents at 4-25°C. After opening, store Reagent A at 4-8°C.
Shelf life
Expiration date is indicated on the box.

Additional items needed

  • LB medium (5 mL), shake flask for pre-culture
  • Sterile solution of 15NH4Cl (40 g/L)
  • Sterile shake flask (5 L) or Ultra Yield Flask (2.5 L)
  • Antibiotics
  • Inducer such as IPTG
  • Anti-foaming agent, such as AntiFoam 204 when using Ultra Yield Flasks or other baffled (fluted) flasks

Note: For shake flask cultures, we recommend the use of Ultra Yield Shake Flasks with AirOtop Enhanced Seals. Improved aeration has been shown to further enhance the performance of EnPresso growth systems for bacterial cultures.

Recommended conditions

  • Culture volume: 10% of flask volume (20% only if using Ultra Yield Flasks)
  • Shaking: 250 rpm, 25-50 mm amplitude. Use 25 mm amplitude for Ultra Yield Flasks.
  • Temperature pre-culture: 37°C
  • Temperature culture: 30°C

It is essential to follow the EnPresso B Defined Nitrogen-free protocol in detail. Using a fresh pre-culture, the correct shake speed, air-permeable closures and recommended cultivation times are critical to ensure success.

Caution: Never use aluminum foil or plastic lids during incubation.

EnPresso B Defined Nitrogen-free 500 – from pre-culture to harvest within 2 days

Protocol for 15N labeling in shake flasks (500 mL culture)

Day 1

  1. Prepare an inoculum from a glycerol stock or use a single colony grown overnight on an agar plate. Inoculate 5 mL of LB medium containing antibiotics in a cultivation tube or shake flask.
  2. Incubate with vigorous shaking at 37°C for 6 h or until the inoculum has reached OD 2 - 4.
  3. Add aseptically into a sterile 5 L shake flask
    - 465mL sterile water
    - 20 tablets (one bag) of EnPresso B Defined Nitrogen-free 500
    - 30 mL 15NH4Cl (40 g/L stock, final concentration 2.5 g/L)

    Note: 2.5 g/L final concentration of 15NH4Cl is recommended as a starting concentration. Concentrations as low as 1.0 g/L and as high as 4.0 g/L have also been successfully used.

  4. Immediately shake vigorously at 37°C until the tablets have fully dissolved. After dissolution, small crystals of magnesium salts may occasionally be visible, but these will not affect performance.
  5. Add required antibiotics. If using Ultra Yield Flasks, add anti-foaming agent, such as 50 μL AntiFoam 204 in 500 mL culture volume.
  6. Inoculate with 1:100 of the pre-culture inoculum (5 mL).
  7. Add 250 μL Reagent A (final concentration 1.5 U/L).
  8. Close the flask securely.
  9. Incubate overnight (16-20 h) at 30°C, 250 rpm.

Day 2

  1. Sample after 6 to 8 hours induction to evaluate protein production.
  2. Continue incubation overnight.
  3. Sample after 6 to 8 hours induction to evaluate protein production.
  4. Continue incubation overnight.

Day 3

  1. Harvest.

Note: This protocol has been optimized to enhance the growth of E. coli. For other tips and hints on how to achieve optimal performance when growing bacteria,



For research use only. Not for human consumption or diagnostic use.

Related Links