Methods of Protein Microcharacterization: A Practical by Petro E. Petrides (auth.), John E. Shively (eds.)

By Petro E. Petrides (auth.), John E. Shively (eds.)

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It may become necessary to replace the column or to use another column brand. Sometimes even columns from the same manufacturer show different recovery properties, which may be because of the utilization of different silica batches for packing. If low recoveries persist, the mobile phase should also be changed. Best recoveries are usually obtained with pyridine formate/npropanol, TFA/acetonitrile, and triethylammonium phosphate/ acetonitrile. 4. Evaluation of Purity of Sample Prior to Sequencing As in classical TLC, the purity of the peptide or small protein samples obtained should be proven by rechromatography in 26 Petrides mobile phases with different solute selectivity.

01 ^1 S p I-, 4j in viH ^ '-O _ . ts 60^^ c ( — ) % 'louDdojd-u 60 ^2 28 Petrides 500 400 80 60- CD O C o ^ • ^ - 300 (/) a> o c o U -^-^ ^3 o «+- Q. a Q. I Li_ O 200 UJ 0) C OC 18 100 12 15 30 45 60 75 Retention time (min) 90 Fig. 7. Rechromatography of immunoreactive material from preparative RPLC. Fractions 73-77 (Fig. 0). The column was eluted with a linear gradient from 14 to 15% n-propanol over 75 min. 0 mL/min. S-jitL aliquots used for the RIA (26). with fluorescamine (lower panel) with the detectors arranged in series at the column exit.

As a result of the addition of fluorescamine, there is a small base-line rise, and Microisolation of Polypeptides 21 the waste from the fluorescamine system starts to smell of acetone. The detection can be started as soon as a steady base-line has been reached. , effective buffering of the acidic RPLC effluent by the borate buffer system can be further ascertained by determination of the pH of the effluent from the fluoromonitor, which should be in the alkaline range. , first the fluorescamine pump is shut off and then—if possible—the slider valve X.

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