Analytical extraction of regulatory peptides from rat lung tissue.
We evaluated protocols for the extraction of calcitonin gene-related peptide, neuropeptide Y, substance P, peptide YY and beta-endorphin from rat lung tissue for subsequent radioimmunoassay. The effects of varying acidity of the extraction solution and repeating extraction on the recovery of peptide immunoreactivity and non-specific tracer-binding were compared by analysis of variance. Moreover, variability of immunoreactivity was quantified for comparison. Considering all three criteria, the optimal acidity for extraction was: 0.1 M or 1 M acetic acid for CGRP and beta-endorphin, 0.1 M acetic acid for NPY, 1 M acetic acid for substance P and phosphate buffer for peptide YY. Double or combined extraction unambiguously improved assay results only for substance P. Reversed-phase high-performance liquid chromatography of CGRP-, NPY- and SP-immunoreactivity obtained from selected extracts suggested that differences in recovery of these peptides are not explainable by differential peptide fragmentation during extraction.
Kraiczi
H
H
Department of Clinical Neuroscience, Sahlgrenska University Hospital/Mölndal, Sweden. holger.kraiczi@pharm.gu.se
Karlsson
G
G
Ekman
R
R
D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000032:analysis / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D008168:Lung / Q000737:chemistry*; D008297:Male; D009478:Neuropeptide Y / Q000032:analysis / Q000302:isolation & purification; D009479:Neuropeptides / Q000032:analysis / Q000302:isolation & purification*; D019894:Peptide YY / Q000032:analysis / Q000302:isolation & purification; D011863:Radioimmunoassay; D051381:Rats; D017207:Rats, Sprague-Dawley; D013373:Substance P / Q000032:analysis / Q000302:isolation & purification; D001615:beta-Endorphin / Q000032:analysis / Q000302:isolation & purification
D009478:Neuropeptide Y; D009479:Neuropeptides; D019894:Peptide YY; D013373:Substance P; D001615:beta-Endorphin; D015740:Calcitonin Gene-Related Peptide