Preparation of samples for hydrolysis

Before the samples are hydrolysed for amino acid analysis, they must be rendered salt-free. Samples are thus dialysed twice against 2 litres of 10 mM sodium chloride in Nanopure water or against a volatile buffer such as 0.5% ammonium bicarbonate. 

Samples are then transferred to new 13 x 100 mm glass culture tubes that have been washed in RBS. 

Samples should contain about 50 to 100 pg protein, equivalent to at least 2 nmol of the least abundant amino acid. 

10 /1 of 1.25 mM norleucine are added to act as an internal standard. After sealing the tubes with parafilm, the samples are frozen in a bath made from solid CO2 carefully added to ethanol after piercing the parafilm a few times with a needle, the samples are left to lyophilize overnight (for volumes of 1 ml and above). 

Small samples may be dried in the liquid state, a procedure which tends to get rid of contaminating ammonia better than lyophilization but runs the risk of sample loss by bumping. Samples can be placed in a desiccator containing a Petri dish of P2O5 (care with water) and evacuated overnight to dry. 

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