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A combined digest using trypsin and endo-GluC yields the fragments of 2,416.9 and 2,430.9 Da that allowed the sequence determination and confirming the difference of a glycine (NTP G) and alanine (NTP A) at the last position of loop 3.
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The combined extracted digest samples were then dried down at the low drying setting (some heat) on a Speed Vac SC110 (Savant Instruments, Holbrook, NY, USA) fitted with a Refrigerated Condensation Trap and a Vac V-500 (Buchi, Flawil, Switzerland).
Heavy and light versions of the reagents are used to label the two different samples before they are combined and digested.
The supernatants and washings were combined and digested with Papain (Sigma-Aldrich) (2.5 mg/ml in 0.1 M Na acetate, 5 mM ethylenediamine tetraacetic acid, pH 6.0) at 65°C for 2 hours.
This solution was combined with digest solution.
To achieve maximum sequence coverage of the protein we combined proteolytic digests from four proteolytic enzymes (trypsin, chymotrypsin, LysC, and subtilisin).
Fragments were combined and double digested with EcoRI/HindIII (NEB) and ligated (Epicenter).
Proteins from each culture are then isolated, combined together, and digested with trypsin to generate peptides.
DNA methylation was determined by digesting Combined Bisulfite and Restriction Analysis (CoBRA) PCR products with the BstUI and TaqI restriction enzymes (Fementas, UK).
The problem of combining restriction digest and sequence information has been studied in the past, albeit from a somewhat different perspective.
For CP, data reported here are in agreement with those of Salem et al. (2006) for different tree foliage digestibility, as well as those of Papachristou (1996), and tend to confirm the apparently greater ability of goats to digest likely combined protein-tannin complexes.
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