Prokaryotic Cell Growth Expression
The most popular medium for bacterial cell growth is minimal medium. The primary reason for the popularity of the use of minimal media is that it remains to be the most economic media available to grow isotope-labeled bacteria. Minimal media is a mixture M9 salts, glucose (as the sole carbon source) and ammonium chloride or ammonium sulfate (as the sole nitrogen source) that is formulated in either H2O or D2O. Often, depending on the E. coli strain used, additional reagents, such as vitamins and cofactors, are added to the media to aid growth and expression.
Alpha Keto Acids (α-Ketoacids)
Fully or partially protonated 13C methyl groups in otherwise per-deuterated proteins are excellent probes for structure and dynamics, and are especially crucial to studying high-molecular weight proteins and protein complexes. The popular ILV labeling strategy utilizes feeding E. coli labeled amino acid precursors to produce protein containing either uniformly or selectively labeled isoleucine, leucine and valine. α-Ketoisobutyrate is the precursor to Ile and may be used to label the δ1 methyl in Ile. α-ketoisovalerate is the precursor to Leu and Val and is used to label both methyl groups in these amino acids (although not in the same molecule). Please see Application Notes 16 and 25 for more information regarding this application.
Methyl labeling Kits
Although methyl labeling of Ile, leu and val is straightforward using alpha keto acids, producing labeled alanine requires additional reagents to suppress metabolic scrambling. Our Alanine and ILVA labeling kits are pre-weighed reagents suitable for addition to 1L of suitably labeled minimal media.
Rich Bacterial Media
Although minimal media offers the most economic means used to label E. coli, the resulting expression levels are sometimes not high enough to conduct the desired studies. An economic method to enhance protein yield and growth characteristics is to spike low levels of suitably labeled rich media into minimal media prior to induction. If this proves insufficient, using rich media full-strength should improve the yield. CIL offers several choices in rich media for labeling E. coli: BioExpress 1000, Celtone Powder, Celtone Complete, and Silantes OD2.
- BioExpresss® 1000
- Celtone® Complete
- Celtone® Powder
- Silantes® E. coli-OD2
- Spectra 9 Media