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Development of Analytical Methods for Determination of Peptide Concentration
Author(s) -
Lang Ewa,
Rupprecht Kevin,
Fishpaugh Jeffrey
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.577.3
Subject(s) - bicinchoninic acid assay , chemistry , peptide , chromatography , lysine , amino acid , histidine , peptide sequence , chromophore , biochemistry , organic chemistry , gene
Measuring the concentration of peptides can be challenging. Amino acid sequence, solubility and the folding of the peptide can influence measurement. A280 measurement requires that chromophores trytophan and tyrosine be in the sequence. Lyophilized peptides contain water and salts so concentration determined by weight alone is imprecise. Amino acid analysis is the default method for measuring peptide concentration, but it is very labor intensive. We evaluated two microtiter plate methods for potential use in peptide quantitation. Pierce BCA Protein Assay (bicinchoninic acid) combines the reduction of Cu2+ to Cu1+ by protein in an alkaline medium with the selective colorimetric detection of the (Cu1+) by bicinchoninic acid. Peptides of three or more residues form the reaction, which is measured at 562 nm. LavaPep™ Protein & Peptide quantification kit depends on a small, naturally‐occurring fluorescent compound, epicocconone, that reversibly binds to lysine, arginine, and histidine residues to yield an intensely red‐fluorescent product (Ex/Em: 540/620 nm). Various peptides were used to generate standard curves to characterize the affect of standards used on the concentration. Results presented will demonstrate the relative robustness, accuracy and precision of the two methods.