Protein Preparation
The protein is produced by expression from bacteria which are grown on minimal medium supplemented with ¹⁵NH₄Cl and either 1,3-¹³C-labelled glycerol or 2-¹³C-labelled glycerol.

The labelling scheme obtained is somewhat involved. In the following figure the sites coloured in blue are labelled in the 1,3-¹³C glycerol sample and the sites coloured in red are labelled in the 2-¹³C glycerol sample.

For those amino acids synthesised via the citric acid cycle several so-called isotopomers are obtained which then result in the average labelling shown above. For threonine the individual isotopomers which give rise to the average labelling are shown. A complete figure of all the isotopomers is available here. Note that the relative populations of isotopomers shown in these figures are those determined for SH3 (Castellani et al. 2002) – for other proteins they may be slightly different.

Applications
Originally labelling with 2-¹³C-glycerol was used to for relaxation measurements in solution (LeMaster and Kushlan, 1996). The Cα atoms in such samples do not contain any neighbouring ¹³C-labelled atoms (with the exception only of Valine). Relaxation measurements on the isolated ¹³Cα-¹Hα groups can therefore be interepreted more easily without having to take couplings to the neighbouring carbon atoms into account.
More recently Castellani et al. (2002) showed that 1,3-¹³C- and 2-¹³C-glycerol labelled samples are useful for protein structure determination in solid-state MAS NMR. The samples have several advantages: (a) many J-couplings are removed which results in sharper lines, (b) very strong dipolar couplings between neighbouring atoms are removed and enable more long-range correlations via weak dipolar couplings to be observerd and (c) the spectral overlap is decreased. Currently this is probably the most common application of 1,3-¹³C- and 2-¹³C-glycerol labelling. The characteristic labelling patterns for each amino acid can also be exploited for assignment purposes (Higman et al. 2009).
Hong and Jakes (1999) combined 2-¹³C-glycerol labelling with amino acid specific labelling in order to label only half the amino acids with 2-¹³C-glycerol.

Variants
See here for a varient in which 1- and 2-¹³C-glucose is used rather than 1,3- and 2-¹³C-glycerol.

References:
D.M. LeMaster and D.M. Kushlan (1996) J. Am. Chem. Soc. 118 9255-9264. (Link to Article)
M. Hong and K. Jakes (1999) J. Biomol. NMR 14 71-74. (Link to Article)
F. Castellani, B.-J. van Rossum, A. Diehl, M. Schubert, K. Rehbein and H. Oschkinat (2002) Nature 420 98-102. (Link to Article)
V. A. Higman, J. Flinders, M. Hiller, S. Jehle, S. Markovic, S. Fiedler, B.-J. van Rossum and H. Oschkinat (2009) J. Biomol. NMR 44 245-260. (Link to Article)