HP 241 N-to-C and C-to-N Column Switching Techniques Guide 5.0

N-to-C 5.0 and C-to-N 5.0 Protein Sequencer Methods Technical Note

HP 241 Protein Sequencer (N+C)

The N-to-C 5.0 and C-to-N 5.0 The C-to-N 5.0 method is run when The methods are: sequencer methods are responsi- switching from the C-terminal to • N-to-C 5.0 (column) ble for the switching between the N-terminal sequencing chem- • N-to-C 5.0 (flask) the N-terminal and C-terminal istry. The column method washes • Column wash 5.0 sequencing chemistry methods the column, while the flask method, • Flask wash 5.0 on the Hewlett-Packard column- in addition to washing the flask, based protein sequencer. prepares a coordinated injection The sequence program controls all

with the on-line HPLC to place the of the column and flask methods,

The N-to-C 5.0 method is run when proper HPLC solvents in the HPLC as well as any cycle exception switching from the N-terminal to binary pumps for the N-terminal methods. The N-to-C 5.0 and the C-terminal sequencing chem- chromatography. These methods C-to-N 5.0 sequencer methods istry. The column method pauses are generally run with a Zitex strip do not use an exception method. the column, while the flask method in an empty RP/SAX biphasic col- prepares a coordinated injection umn, but any column configuration with the on-line HPLC to place the is applicable. No column prepara- proper HPLC solvents in the HPLC tion is required with this method. binary pumps for the C-terminal chromatography.

The Routine 5.0 sequencer methods require the bottle configuration shown. The reagents and solvents are purchased from Hewlett-Packard. These methods produce less than 1m L/cycle of liquid waste.

S2A Neat ethyl acetate 1R Diphenylphosphorylisothiocyanate (DPP-ITC) in toluene/heptane (23:27:50) R1 Phenylisothiocyanate (PITC) in heptane (3:97) 2R Pyridine in ethylacetate (2:98) R2 Diisopropylethylamine (DIEA) in 1-propanol/water (1:6:3) S2/3 Acetonitrile/toluene (23:77) R2A Ocylamine in heptane (3:97) 3R Potassium trimethylsilanolate (0.1M) in methanol/t-butanol (50:50) S3 Acetonitrile/toluene (15:85) R3 Neat trifluoroacetic acid (TFA) L3 Acetic acid in methanol/water (1:74:25) PTH Mixture of PTH amino acids (10 pmol/100 µl) in acetonitrile with DPTU

as a marker

TH Mixture of TH amino acids (50 pmol/100 µl) in acetonitrile R4 Trifluoroacetic acid (TFA) in water (1:3) S5 Phosphate buffer (p H 2.9), 0.2% ion pairing reagent