Attachment IV - Page 31 
-9- DRAFT 
NOVEMBER 7, 1983 
peptide napping can provide precise evidence for the identity of a protein. For 
proteins containing disulfide bonds, peptide napping can be used to verify the 
proper arrangement of disulfide bonds in the final product. 
A. Polyacrylamide Gel Electrophoresis (PAGE) and Isoelectric Focusing 
PAGE and Isoelectric focusing are valuable techniques for verifying 
the purity and molecular weight of proteins and peptides. The PA GE gels should be 
run in sodium dodecylsulfate with and without exposure to reducing agents, and 
with appropriate molecular weight standards or reference preparations. 
It is preferable to analyze duplicate samples on slab gels, one 
stained with Coomassie blue and the other stained with silver. The silver stain 
is considerably more sensitive for the detection of very small quantities of 
proteins and is useful in identifying nonprotein materials such as nucleic acid, 
carbohydrate and lipid which may be present. 
For peptides of molecular weight less than ca. 8,000 daltons, most 
PAGE methods may not be accurate for molecular weight estimates. 
5. High Performance Liquid Chromatography (HPLC) 
HPLC is a very useful method to determine the purity of a protein 
or peptide, to evaluate its molecular configuration and, under some circumstances, 
to confirm its identity. HPLC may be especially useful in characterizing and 
quantitating specific impurities in the final product. A widely used method is 
reverse-phase HPLC. A protein or peptide which elutes as a single symmetrical 
peak, in two markedly different systems, Including an ion-pair system, is generally 
quite pure. 
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