Below is the method that we advise for Sample Preparation of a Protein Hydrolysate Sample and has been taken from the Biochrom 20 manual.
Before any protein or polypeptide can be subjected to amino acid analysis, the sample must be hydrolysed to cleave the peptide bonds between the amino acids. Acid hydrolysis using hydrochloric acid is the most common hydrolysis method in use, however, the severe conditions of this method can partially or totally destroy some amino acids. Some amino acids are difficult to cleave and require different conditions to achieve complete hydrolysis.
Many different methods of hydrolysis are available, other than the acid hydrolysis described below, these methods being designed to provide better recovery of specific amino acids. Other hydrolysis methods are used to cleave the peptide bonds of resistant amino acids and also to cleave amino acids in the presence of concentrations of carbohydrates or fats which would interfere with acid hydrolysis. Further information is available on request from support@biochrom.co.uk.
To perform an acid hydrolysis proceed as follows:
Place a known amount of sample into a thick walled glass hydrolysis tube.
Add an aliquot of constant boiling (5.7M) hydrochloric acid to the hydrolysis tube in the ratio of 1ml of hydrochloric acid to 10mg of protein.
Attach the hydrolysis tube to a system which allows the connection of nitrogen and vacuum lines without disturbing the sample.
Evacuate the sample to remove the air then purge the sample with oxygen free nitrogen. Completely evacuate the tube before sealing it. It may be necessary to freeze the sample to prevent boiling during evacuation.
Heat the sealed tube, in an oven, to 110°C for 24 hours.
Remove the tube from the oven and extract the acid from the sample by lyophilisation, rotary evaporation or drying down over sodium hydroxide, under vacuum.
Dilute the sample using a known amount of pH 2.2 loading buffer and filter the sample using a 0.2µm membrane (e.g. Millipore or Nucleopore).
The hydrochloric acid used in this hydrolysis must be pure and of constant boiling fraction.
If this does not answer your question or solve your problem, then please contact us at support@biochrom.co.uk detailing your question or the problem you are experiencing. Please list if relevant the instrument, software and version numbers you are running, and enclose any relevant data files, etc.