Amino Acid Analysis Protocols (Methods in Molecular Biology by Cooper C., Packer N.

By Cooper C., Packer N.

Proteome structures Ltd, North Ryde, Australia. Describes quite a few amino acid research suggestions and the way every one process can be utilized to reply to particular biologic questions.

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Trifluoroacetic acid (TFA). Methanol. Hydrochloric acid (HCl). Phenol. 3. Reagents 1. 1 N HCl. 2. 1% phenol. 3. 1% TFA. 3. 1. Preparation of the Macrospin column (sample 50–150 µ L) 1. The macrospin columns should be prepared basically as suggested by the manufacturer (2), except that we have introduced several more washes to better equilibrate the columns. In brief, tap the column gently to recover the gel material at the bottom of the column. Hydrate the gel with 500 µL of HPLC-grade water for 15 min at room temperature (see Note 6) and then centrifuge the column for 4 min at about 2000g to equilibrate the column.

4 (see Note 20). 5. Carboxypeptidase Analysis Applications involving single or combinations of carboxypeptidases to assign C-terminal protein sequences have been adequately described elsewhere (18). 1. , 10 nmol Nle for a sample containing 10 nmol of polypeptide). 2. Take aliquots at various time-points and place in Eppendorf tubes containing an equal volume of 2 N glacial acetic acid. 3. Heat for 2 min at 100°C on a boiling water bath to halt the digestion and precipitate the protein. Role of AAA in a Biotechnology Laboratory 19 E 66 = E40 = IF(E41/[H$62]=0, " " = IF(E42/[H$62]=0, " " = IF(E43/[H$62]=0, " " = IF(E44/[H$62]=0, " " = IF(E45/[H$62]=0, " " = IF(E46/[H$62]=0, " " = IF(E47/[H$62]=0, " " = IF(E48/[H$62]=0, " " = IF(E49/[H$62]=0, " " = IF(E50/[H$62]=0, " " = IF(E51/[H$62]=0, " " = IF(E52/[H$62]=0, " " = IF(E53/[H$62]=0, " " = IF(E54/[H$62]=0, " " = IF(E55/[H$62]=0, " " = IF(E56/[H$62]=0, " " = IF(E57/[H$62]=0, " " = IF(E58/[H$62]=0, " " = IF(E59/[H$62]=0, " " = IF(E60/[H$62]=0, " " ,[E41/H$62]) ,[E42/H$62]) ,[E43/H$62]) ,[E44/H$62]) ,[E45/H$62]) ,[E46/H$62]) ,[E47/H$62]) ,[E48/H$62]) ,[E49/H$62]) ,[E50/H$62]) ,[E51/H$62]) ,[E52/H$62]) ,[E53/H$62]) ,[E54/H$62]) ,[E55/H$62]) ,[E56/H$62]) ,[E57/H$62]) ,[E58/H$62]) ,[E59/H$62]) ,[E60/H$62]) 4.

Derivatization Reaction 1. Add 50 µL of triethylamine/methanol/water (1:1:2) to the dried sample vials, mix vigorously by vortexing, and dry them under reduced pressure (see Note 2). 2. 4 mM) by dissolving 5 mg of Marfey’s reagent in 1 mL of acetone. 3. Dissolve the dried amino acid mixture or the hydrolyzed protein sample in 100 µL of 25% (v/v) triethylamine, add 100 µL of the Marfey’s reagent solution, and mix by vortexing (see Note 3). 4. Incubate the reaction vial at 40°C for 60 min with gentle shaking protected from light (see Note 4).

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